Novel forms of plant defensins

ABSTRACT

The present invention relates to heterogeneous or artificially created forms of plant defensins, and to uses of such heterogenous or artificially created defensins including methods for preventing or treating proliferative diseases. Compositions for use as animal and human medicaments are also provided. The present invention also relates to associated methods, uses, systems and kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/548,825 filed on 19 Oct. 2011, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to heterogeneous forms of defensins, andto uses of such heterogeneous defensins including methods for preventingor treating proliferative diseases. Animal and human medicaments arealso provided. The present invention also relates to associated methods,uses, systems and kits.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

Not applicable.

BACKGROUND TO THE INVENTION

Plants are known to produce a variety of chemical compounds, eitherconstitutively or inducibly, to protect themselves against environmentalstresses, wounding, or microbial invasion.

Of the plant antimicrobial proteins that have been characterized todate, a large proportion share common characteristics. They aregenerally small (<10 kDa), highly basic proteins and often contain aneven number of cysteine residues (typically 4, 6 or 8). These cysteinesall participate in intramolecular disulfide bonds and provide theprotein with structural and thermodynamic stability (Broekaert et al.(1997)). Based on amino acid sequence identities, primarily withreference to the number and spacing of the cysteine residues, a numberof distinct families have been defined. They include the plant defensins(Broekaert et al., 1995, 1997; Lay et al., 2003a), thionins (Bohlmann,1994), lipid transfer proteins (Kader, 1996, 1997), hevein (Broekaert atal., 1992) and knottin-type proteins (Cammue et al., 1992), as well asantimicrobial proteins from Macadamia integrifolia (Marcus et al., 1997;McManus at al., 1999) and Impatiens balsamina (Tailor et al., 1997;Patel at al., 1998) (Table 1). All these antimicrobial proteins appearto exert their activities at the level of the plasma membrane of thetarget microorganisms, although it is likely that the different proteinfamilies act via different mechanisms (Broekaert et al., 1997). Thecyclotides are a new family of small, cysteine-rich plant peptides thatare common in members of the Rubiaceae and Violaceae families (reviewedin Craik et al., 1999, 2004; Craik, 2001). These unusual cyclic peptides(Table 1) have been ascribed various biological activities includingantibacterial (Tam, et al., 1999), anti-HIV (Gustafson et al., 1994) andinsecticidal (Jennings et al., 2001) properties.

TABLE 1 Small, cysteine-rich antimicrobial proteins in plants. Repre-No. of Peptide sentative amino family member acids Consensus sequencePlant defensins Rs-AFP2 51

α/β-Thionin (8-Cys type) α- Purothionin 45

Lipid transfer protein Ace-AMPI 93

Hevein- type Ac-AMP2 30

Knottin- type Mj-AMP1 36

Macadamia MiAMP1 76

Impatiens Ib-AMP1 20

Cyclotide Kalata B1 29

The size of the mature protein and spacing of cysteine residues forrepresentative members of plant antimicrobial proteins is shown inTable 1. The numbers in the consensus sequence represent the number ofamino acids between the highly conserved cysteine residues in therepresentative member, but other members of the family may vary slightlyin the inter-cysteine lengths. The disulfide connectivities are given byconnecting lines. The cyclic backbone of the cyclotides is depicted bythe broken line (from Lay and Anderson, 2005).

Defensins

The term “defensin” has previously been used in the art to describe adiverse family of molecules that are produced by many different speciesand which function in innate defense, against pathogens includingbacteria, fungi, yeast and viruses.

Plant Defensins

Plant defensins (also termed γ-thionins) are small (˜5 kDa, 45 to 54amino acids), basic proteins with eight invariant cysteine residues thatform four strictly conserved disulfide bonds with a Cys_(I)-Cys_(VIII),Cys_(II)-Cys_(IV), Cys_(III)-Cys_(VI) and Cys_(V)-Cys_(VII)configuration. As well as these four strictly conserved disulfide bonds,some plant defensins have an additional disulfide bond (Lay et al.,2003a, 2003b; Janssen et al., 2003).

The name “plant defensin” was coined in 1995 by Terras and colleagueswho isolated two antifungal proteins from radish seeds (R5-AFP1 andR5-AFP2) and noted that at a primary and three-dimensional structurallevel these proteins were distinct from the plant α-/β-thionins butshared some structural similarities to insect and mammalian defensins(Terras et al., 1995; Broekaert et al., 1995).

Plant defensins exhibit clear, although relatively limited, sequenceconservation. Strictly conserved are the eight invariant cysteineresidues and a glycine at position 32 (numbering relative to thecontinuous NaD1 sequence; see for example SEQ ID NO: 22 and the sequencealignments shown in any of FIGS. 11 to 15) or position 34 (numberingrelative to R5-AFP2). With reference to the numbering of amino acidsresidues in R5-AFP2, in most of the sequences a serine at position 8(position 7 in NaD1), an aromatic residue at position 11 (position 10 inNaD1), a glycine at position 13 (position 12 in NaD1) and a glutamicacid at position 29 (position 27 in NaD1) are also conserved (Lay etal., 2003a; Lay and Anderson, 2005).

The three-dimensional solution structures of the first plant defensinswere elucidated in 1993 by Bruix and colleagues for γ1-P and γ1-H (alsoreferred to herein as “g1-H”). Since that time, the structures of otherseed-derived and two flower-derived (NaD1 and PhD1) defensins have beendetermined (Lay et al., 2003b; Janssen et al., 2003). All thesedefensins elaborate a motif known as the cysteine-stabilized αβ (CSαβ)fold and share highly superimposable three-dimensional structures thatcomprise a well-defined α-helix and a triple-stranded antiparallelβ-sheet. These elements are organized in a βαββ arrangement and arereinforced by four disulfide bridges.

The CSαβ motif is also displayed by insect defensins and scorpiontoxins. In comparing the amino acid sequences of the structurallycharacterized plant defensins, insect defensins and scorpion toxins, itis apparent that the CSαβ scaffold is highly permissive to size andcompositional differences.

The plant defensin/γ-thionin structure contrasts to that which isadopted by the α- and β-thionins. The α- and β-thionins form compact,amphipathic, L-shaped molecules where the long vertical arm of the L iscomposed of two α-helices, and the short arm is formed by twoantiparallel β-strands and the last (˜10) C-terminal residues. Theseproteins are also stabilized by three or four disulfide bonds (Bohlmannand Apel, 1991).

Plant defensins have a widespread distribution throughout the plantkingdom and are likely to be present in most, if not all, plants. Mostplant defensins have been isolated from seeds where they are abundantand have been characterized at the molecular, biochemical and structurallevels (Broekaert et al., 1995; Thomma et al., 2003; Lay and Anderson,2005). Defensins have also been identified in other tissues includingleaves, pods, tubers, fruit, roots, bark and floral tissues (Lay andAnderson, 2005).

An amino acid sequence alignment of several defensins that have beenidentified, either as purified protein or deduced from cDNAs, has beenpublished by Lay and Anderson (2005). Other plant defensins have beendisclosed in U.S. Pat. No. 6,911,577, International Patent PublicationNo. WO 00/11196 and International Patent Publication No. WO 00/68405,the entire contents of which are incorporated herein by reference.

Two Classes of Plant Defensins

In the first and largest class, Class I, the precursor protein iscomposed of an endoplasmic reticulum (ER) signal sequence and a maturedefensin domain. These proteins enter the secretory pathway and have noobvious signals for post-translational modification or subcellulartargeting (FIG. 10A).

The second class of defensins are produced as larger precursors withC-terminal prodomains or propeptides (CTPPs) of about 33 amino acids(FIG. 10B). Class II defensins have been identified in solanaceousspecies where they are expressed constitutively in floral tissues (Layet al., 2003a; Gu et al., 1992; Milligan et al., 1995; Brandstadter etal., 1996) and fruit (Aluru et al., 1999) and in salt stressed leaves(Komori et al., 1997; Yamada et al., 1997). The CTPP of the solanaceousdefensins from Nicotiana alata (NaD1) and Petunia hybrida (PhD1 andPhD2) is removed proteolytically during maturation (Lay et al., 2003a).

Biological Activity of Plant Defensins

The best characterized activity of some but not all plant defensins istheir ability to inhibit, with varying potencies, a number of fungalspecies (for examples, see Broekaert et al., 1997; Lay et al., 2003a;Osborn et al., 1995). Growth inhibitory effects on Gram-positive andGram-negative bacteria have also been described (Segura et al., 1998;Moreno et al., 1994; Zhang and Lewis, 1997).

The inventors have previously disclosed in International PatentPublication No. WO 02/063011 certain defensins and their use in inducingresistance in plants or parts of plants to pathogen infestation. Theentire content of WO 02/063011 is incorporated herein by reference.

Plant Defensins with Antiproliferative Activity in Cancer Cells

The inventors have previously disclosed that Solanaceous Class II plantdefensins have activity in preventing or treating proliferative diseasesin mammals. Methods of treating or preventing proliferative disease byadministering plant defensins, together with associated uses, kits andpharmaceutical compositions, are disclosed in international patentapplication no. PCT/AU2011/000760 filed on 23 Jun. 2011. Such methods,uses, kits and pharmaceutical compositions are also disclosed in U.S.patent application Ser. No. 13/166,960, also filed on 23 Jun. 2011. Theentire content of both PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 isincorporated herein by reference.

Previous attempts to modify the defensins to improve and broaden theiractivity have hitherto been largely unsuccessful. Thus, despite theutility of the inventions disclosed in WO 02/063011, PCT/AU2011/000760and U.S. Ser. No. 13/166,960 there remains a great need in the relevantart for plant defensins that display enhanced anti-proliferativeactivity and/or broader spectrum of activity.

Further studies by the inventors into the mechanism of action of plantdefensins by the inventors have surprisingly revealed that Class IISolanaceous plant defensins, such as NaD1 may target cells through therecognition of a ‘phospholipid pattern’ by a specific region of thedefensin sequence.

This information provides basis for developing new defensins withadvantageous qualities and functions, including but not limited tofunctionalisation of defensins that are inactive or have low activity inrelation to treatment of proliferative diseases, variation of, oraddition of new, plant defensin effector functions, and development ofdefensins with enhanced cytotoxic potency. These new plant defensinstherefore also provide for new methods of treatment, uses and kitssuitable for the prevention or treatment of diseases, disorders andailments such as proliferative diseases.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided aheterogeneous plant defensin, wherein the plant defensin comprises afirst polypeptide sequence and a second polypeptide sequence, whereinthe second polypeptide sequence is derived from a plant defensin otherthan the plant defensin from which the first polypeptide sequence isderived.

In some embodiments, the heterogeneous plant defensin comprises a thirdpolypeptide sequence.

In some embodiments, the heterogeneous plant defensin contains eightinvariant cysteine amino acids.

In some embodiments, the heterogeneous plant defensin contains at leastfive loop regions.

In some embodiments, the heterogeneous plant defensin comprises thefollowing amino acid sequence:

(SEQ ID NO: 1)X_(A)-C1-X_(B)-C2-X_(C)-C3-X_(D)-C4-X_(E)-C5-X_(F)-C6-X_(G)-C7-X_(H)-C8-X_(I);wherein C1, C2, C3, C4, C5, C6, C7 and C8 is cysteine, said cysteinebeing an invariant cysteine, X_(A), X_(B), X_(C), X_(D), X_(E), X_(F),X_(G), X_(H), X_(I) is any naturally or non-naturally occurring aminoacid and;X_(A) is 1 to 7 amino acids in length;X_(B) is 9, 10 or 11 amino acids in length;X_(C) is 3 to 8 amino acids in length;X_(D) is 3 amino acids in length;X_(E) is 9 to 13 amino acids in length;X_(F) is 4, 5, 6, 7 or 8 amino acids in length;X_(G) is 1 amino acid in length;X_(H) is 1 to 4-amino acids in length; andX_(I) is 0 or 1 amino acid in length.

In some embodiments, the heterogeneous plant defensin comprises thefollowing amino acid sequence:

(X)¹⁻⁷-C-(X)³⁻⁴-S-(X)₂-o-(X)₁-g-(X)₁-C-(X)³⁻⁸-C-(X)₃-C-(X)²⁻⁴-e-(X)⁴⁻⁶-g-(X)₁-C-(X)⁴⁻⁸-C-(X)₁-C- (X)¹⁻⁴-C-(X)⁰⁻¹wherein C is cysteine, said cysteine being an invariant cysteine; S isserine, o represents an aromatic amino acid (phenylalanine, tryosine,tryptophan or histidine), e represents glutamate, g represents glycineand (X)_(n) represents any naturally occurring or non-naturallyoccurring amino acid and the integer represents the number of aminoacids in that may comprise the specific region of the amino acidsequence.

In some embodiments, the second polypeptide sequence is positionedbetween the fourth and eighth invariant cysteine residues.

In some embodiments, the second polypeptide is positioned between thefifth and sixth invariant cysteine residues.

In preferred embodiments the first polypeptide sequence is derived froma Class I plant defensin.

In preferred embodiments the second polypeptide sequence is derived froma Class II Solanaceous plant defensin.

In certain embodiments, the first polypeptide sequence is derived from aClass I plant defensin and the second polypeptide sequence is derivedfrom a Class II Solanaceous plant defensin.

In some embodiments the third polypeptide sequence is derived from aClass I plant defensin.

In some embodiments, the second polypeptide sequence comprises a loop 5region derived from a Class II Solanaceous plant defensin, or a fragmentor variant thereof.

In some embodiments, the loop 5 region comprises the entire loop 5region of a Class II Solanaceous plant defensin, said loop 5 defined asby being flanked by the fifth and sixth invariant cysteine (Cys) aminoacid residues of a Class II Solanaceous plant defensin amino acidsequence.

In certain embodiments, the loop 5 region of the heterogeneous plantdefensin may begin with a serine amino acid positioned adjacent to thefifth invariant cysteine amino acid residue, wherein the serine aminoacid is positioned to the C-terminal side of the fifth invariantcysteine residue.

In certain embodiments, loop 5 region comprises an amino acid sequencebeginning at the end of the second β-strand and ending at the N-terminalside of the sixth invariant cysteine amino acid residue of a Class IISolanaceous plant defensin amino acid sequence.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serineor arginine, X₂ is lysine, arginine or histidine, X₃ and X₄ are each ahydrophobic amino acid, X₅ is arginine, lysine or histidine and X₆ isarginine, lysine, histidine or asparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serineor arginine, X₂ is lysine, X₃ is isoleucine, leucine or valine, X₄ isleucine or glutamine, X₅ is arginine and X₆ is arginine, lysine orasparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine,arginine or histidine, X₃ and X₄ are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆ is arginine, lysine, histidineor asparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, X₃is isoleucine, leucine or valine, X₄ is leucine or glutamine, X₅ isarginine and X₆ is arginine, lysine or asparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence selected from the group comprising (i) SKILRR (SEQID NO: 2), (ii) SKLLRR (SEQ ID NO: 4), (iii) SKILRK (SEQ ID NO: 6), (iv)SKVLRR (SEQ ID NO: 8), (v) SKVLRK (SEQ ID NO: 10), (vi) SKLQRK (SEQ IDNO: 12), (vii) SKLLRN (SEQ ID NO: 14), (viii) SKLLRK (SEQ ID NO: 16),(ix) SKIQRN (SEQ ID NO: 18), (x) RKLQRK (SEQ ID NO: 20) or (xi) KILRR(SEQ ID NO: 89), (xii) KLLRR (SEQ ID NO: 91), (xiii) KILRK (SEQ ID NO:93), (xiv) KVLRR (SEQ ID NO: 95), (xv) KVLRK (SEQ ID NO: 97), (xvi)KLQRK (SEQ ID NO: 99), (xvii) KLLRN (SEQ ID NO: 101), (xviii) KLLRK (SEQID NO: 103), (xix) KIQRN (SEQ ID NO: 105) or (xx) KLQRK (SEQ ID NO:107).

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising S-K-I-L-R-R (SEQ ID NO:2).

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising K-I-L-R-R (SEQ ID NO:89).

In some embodiments, the first and the third polypeptide sequences areboth derived from the same plant defensin.

In some embodiments, the first polypeptide sequence and optionally thethird polypeptide sequence contain one or more amino acid substitutions,deletions or modifications, wherein the substitution, deletion ormodification amino acid does not naturally occur at the substituted,deleted or modified position in the plant defensin from which the firstpolypeptide sequence and optionally the third polypeptide sequence isderived.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region, said loop 5 region beingpositioned between the fifth and sixth invariant cysteine residues ofthe heterogeneous plant defensin amino acid sequence, and wherein thesecond polypeptide sequence corresponds to an amino acid sequencecomprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serine or arginine, X₂ islysine, arginine or histidine, X₃ and X₄ are each a hydrophobic aminoacid, X₅ is arginine, lysine or histidine and X₆ is arginine, lysine,histidine or asparagine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region, said loop 5 region beingpositioned between the fifth and sixth invariant cysteine residues ofthe heterogeneous plant defensin amino acid sequence, and wherein thesecond polypeptide sequence corresponds to an amino acid sequencecomprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serine or arginine, X₂ islysine, X₃ is isoleucine, leucine or valine, X₄ is leucine or glutamine,X₅ is arginine or lysine and X₆ is arginine or lysine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least invariant eight cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region of the said loop 5 regionbeing positioned between the fifth and sixth invariant cysteine aminoacid residues of the heterogeneous plant defensin amino acid sequence,and wherein the second polypeptide sequence corresponds to an amino acidsequence comprising X₁-X₂-X₃-X₁-X₅-X₆, wherein X₁ is serine or arginine,X₂ is lysine, X₃ is isoleucine, X₁ is leucine, X₅ is arginine and X₆ isarginine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region positioned between thefifth and sixth invariant cysteine residues, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, arginine or histidine, X₃ and X₄are each a hydrophobic amino acid, X₅ is arginine, lysine or histidineand X₆ is arginine, lysine, histidine or asparagine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region positioned between thefifth and sixth invariant cysteine residues, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, X₃ is isoleucine, leucine orvaline, X₄ is leucine or glutamine, X₅ is arginine or lysine and X₆ isarginine or lysine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region positioned between thefifth and sixth invariant cysteine residues, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, X₃ is isoleucine, X₄ is leucine,X₅ is arginine and X₆ is arginine.

In particular embodiments, the heterogeneous plant defensin is selectedfrom SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO:65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ IDNO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQID NO: 85, SEQ ID NO: 87, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ IDNO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88

In certain embodiments, the heterogeneous plant defensin has enhancedand/or broader spectrum anti-proliferative disease activity, or enhancedand/or broader spectrum cytotoxic activity relative to the defensin fromwhich the first polypeptide sequence is derived.

In particular embodiments, there is provided a heterogeneous plantdefensin comprising an amino acid backbone derived from or correspondingto a Class I defensin having at least eight invariant cysteine aminoacid residues and comprising a loop region located between the fifth andsixth invariant cysteine residues, the loop region on the backbone beingsubjected to one or more of a substitution, addition and/or deletionand/or replacement by a loop region, or modified form thereof, from aClass II Solanaceous plant defensin, said Class II Solanaceous plantdefensin having at least eight invariant cysteine amino acid residuesand wherein the substitute loop from the Class II Solanaceous plantdefensin is derived from the region between the fifth and sixthinvariant cysteine residues of the Class II Solanaceous plant defensin,wherein the heterogeneous defensin exhibits enhanced anti-proliferativeactivity and/or broader spectrum of activity compared to the Class Idefensin used as the backbone or the Class II Solanaceous plant defensinfrom which the substitute loop is derived.

In some embodiments the heterogeneous defensin binds to a phospholipid,preferably PIP2 (phosphatidylinositol 4,5-bisphosphate orPtdlns(4,5)P2).

In certain embodiments the second polypeptide sequence, or part thereof,binds to PIP2.

In some embodiments the PIP2 is located in a cell membrane, where saidcell membrane is a tumour or cancer cell membrane.

In particular embodiments the heterogeneous defensin has cell membranepermeabilisation activity.

In a second aspect of the present invention, there is provided a nucleicacid encoding the plant defensin of the first aspect.

In a third aspect of the present invention, there is provided a vectorcomprising the nucleic acid of the second aspect.

In a fourth aspect of the present invention, there is provided a hostcell comprising the vector of the third aspect.

In a fifth aspect of the present invention, there is provided a plantdefensin produced by the host cell of the fourth aspect.

In a sixth aspect of the present invention, there is provided apharmaceutical composition for use in preventing or treating aproliferative disease, wherein the pharmaceutical composition comprisesthe plant defensin of the first aspect, the nucleic acid of the secondaspect, the vector of the third aspect, the host cell of the fourthaspect or the expression product of the fifth aspect, together with apharmaceutically acceptable carrier, diluent or excipient.

In a seventh aspect of the present invention, there is provided a methodfor preventing or treating a proliferative disease, wherein the methodcomprises administering to a subject a therapeutically effective amountof the plant defensin of the first aspect, the nucleic acid of thesecond aspect, the vector of the third aspect, the host cell of thefourth aspect, the expression product of the fifth aspect or thepharmaceutical composition of the sixth aspect, thereby preventing ortreating the proliferative disease.

In an eighth aspect of the present invention, there is provided use ofthe plant defensin of the first aspect, the nucleic acid of the secondaspect, the vector of the third aspect, the host cell of the fourthaspect, the expression product of the fifth aspect or the pharmaceuticalcomposition of the sixth aspect in the preparation of a medicament forpreventing or treating a proliferative disease.

In a ninth aspect of the present invention, there is provided a kit forpreventing or treating a proliferative disease, wherein the kitcomprises a therapeutically effective amount of the plant defensin ofthe first aspect, the nucleic acid of the second aspect, the vector ofthe third aspect, the host cell of the fourth aspect, the expressionproduct of the fifth aspect or the pharmaceutical composition of thesixth aspect.

In a tenth aspect of the present invention, there is provided use of thekit of the ninth aspect for preventing or treating a proliferativedisease, wherein the therapeutically effective amount of the plantdefensin of the first aspect, the nucleic acid of the second aspect, thevector of the third aspect, the host cell of the fourth aspect, theexpression product of the fifth aspect or the pharmaceutical compositionof the sixth aspect is administered to a subject, thereby preventing ortreating the proliferative disease.

DEFINITIONS

The term “derivable” includes, and may be used interchangeably with, theterms “obtainable” and “isolatable”. Compositions or other matter of thepresent invention that is “derivable”, “obtainable” or “isolatable” froma particular source or process include not only compositions or othermatter derived, obtained or isolated from that source or process, butalso the same compositions or matter however sourced or produced.

The term “derived” includes, and may be used interchangeably with, theterms “obtained” and “isolated”. Compositions or other matter of thepresent invention that is “derived” from a particular source or processinclude not only compositions or other matter derived directly from thatsource or process, but also the same compositions or matter derivedindirectly, for example, by way of recombinant DNA technology. In thecase of heterogeneous plant defensins comprising two or morepolypeptides, each polypeptide may be correctly described as “derived”from a particular plant defensin if that polypeptide has been expressedby an expression vector or cassette, such expression vector or cassettehaving had inserted into it a cloned version of the polypeptide.Accordingly, a polypeptide that is “derived” from a plant defensin neednot be the actual polypeptide sourced directly from that plant defensin,but may be an expressed clone of a polypeptide sourced from a plantdefensin.

The terms “heterogeneous”, “heterogeneous plant defensin”,“heterogeneous defensin” “modified plant defensin” or “modifieddefensin” and related terms may be used interchangeably and as usedherein refer to a defensin amino acid or polypeptide sequence that hasbeen modified by the introduction, addition, deletion or substitutionof, for example, one or more naturally or non-naturally occurring aminoacids, polynucleotides or polypeptides. It is to be understood that theamino acids, polynucleotides or polypeptides that may be introduced,added or substituted to produce the heterogeneous defensin, can bederived or obtained or are obtainable from, for example, a defensin suchas a Class II Solanaceous plant defensin. For example, a Class I plantdefensin may be used as a backbone wherein the loop region between fifthand sixth invariant cysteines of the backbone is modified by an aminoacid substitution, addition and/or deletion or substitution or additionof a loop region that is derived from the region between the fifth andsixth invariant cysteines of a Class II Solanaceous defensin, or amodified form thereof, to replace all or part of this loop region in theClass I defensin sequence. The backbone Class I defensin may alsooptionally comprise additional mutations outside this loop region. Whenpresent, from 1 to about 50 additional mutations in the form of an aminoacid substitution, addition and/or deletion may be present in thebackbone Class I defensin.

The terms “enhanced” and “improved” and related terms in the context ofthe invention mean a quantitative or qualitative change, improvement,modulation, increase or modification, in one or more ofanti-proliferative disease activity, cytotoxic activity, spectrum ofactivity, stability and/or membrane permeabilization capacity asrelative a naturally occurring plant defensin or the plant defensinwhich is the subject of modification or relative to the one or moredefensins from which the sequences for the hetereogenous defensin arederived. “Enhancing” and similar terms are intended to encompass anyincrease, change, modification or modulation of anti-proliferativedisease activity, cytotoxic activity or spectrum of activity, stability,solubility and/or membrane permeabilization capacity, whether broughtabout by increase in the activity of the defensin itself, or by increasein the amount of defensin present, or both.

The term “wild-type” as used herein in relation to defensins includespolypeptides in their native form.

As used herein the term “polypeptide” means a polymer made up of aminoacids linked together by peptide bonds, and includes fragments oranalogues thereof. The terms “polypeptide”, “protein” and “amino acid”are used interchangeably herein, although for the purposes of thepresent invention a “polypeptide” may constitute a portion of a fulllength protein.

The term “nucleic acid” as used herein refers to a single- ordouble-stranded polymer of deoxyribonucleotide, ribonucleotide bases orknown analogues of natural nucleotides, or mixtures thereof. The termincludes reference to the specified sequence as well as to the sequencecomplementary thereto, unless otherwise indicated. The terms “nucleicacid”, “polynucleotide” and “nucleotide sequence” are used hereininterchangeably. It will be understood that “5′ end” as used herein inrelation to a nucleic acid corresponds to the N-terminus of the encodedpolypeptide and “3′ end” corresponds to the C-terminus of the encodedpolypeptide.

The term “purified” means that the material in question has been removedfrom its natural environment or host, and associated impurities reducedor eliminated such that the molecule in question is the predominantspecies present. The term “purified” therefore means that an objectspecies is the predominant species present (ie., on a molar basis it ismore abundant than any other individual species in the composition), andpreferably a substantially purified fraction is a composition whereinthe object species comprises at least about 30 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. The terms “purified” and “isolated” may be usedinterchangeably. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A protein ornucleic acid that is the predominant species present in a preparation issubstantially purified. The term “purified” in some embodiments denotesthat a protein or nucleic acid gives rise to essentially one band in anelectrophoretic gel.

The term “fragment” refers to a polypeptide or nucleic acid that encodesa constituent or is a constituent of a polypeptide or nucleic acid ofthe invention thereof. Typically the fragment possesses qualitativebiological activity in common with the polypeptide or nucleic acid ofwhich it is a constituent. A peptide fragment may be between about 5 toabout 150 amino acids in length, between about 5 to about 100 aminoacids in length, between about 5 to about 50 amino acids in length, orbetween about 5 to about 25 amino acids in length. Alternatively, thepeptide fragment may be between about 5 to about 15 amino acids inlength. The term “fragment” therefore includes a polypeptide that is aconstituent of a full-length plant defensin polypeptide and possessesqualitative biological activity in common with a full-length plantdefensin polypeptide. A fragment may be derived from a full-length plantdefensin polypeptide or alternatively may be synthesised by some othermeans, for example chemical synthesis.

The term “fragment” may also refer to a nucleic acid that encodes aconstituent or is a constituent of a polynucleotide of the invention.Fragments of a nucleic acid do not necessarily need to encodepolypeptides which retain biological activity. Rather the fragment may,for example, be useful as a hybridization probe or PCR primer. Thefragment may be derived from a polynucleotide of the invention oralternatively may be synthesized by some other means, for examplechemical synthesis. Nucleic acids of the present invention and fragmentsthereof may also be used in the production of antisense molecules usingtechniques known to those skilled in the art.

The term “recombinant” when used with reference, for example, to a cell,nucleic acid, protein or vector, indicates that the cell, nucleic acid,protein or vector has been modified by the introduction of aheterologous nucleic acid or protein or by the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Accordingly, “recombinant” cells express genes that are notfound within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all. By the term “recombinant nucleic acid” is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid, for example, using polymerases andendonucleases, in a form not normally found in nature. In this manner,operable linkage of different sequences is achieved. Thus an isolatednucleic acid, in a linear form, or an expression vector formed in vitroby ligating DNA molecules that are not normally joined, are bothconsidered “recombinant” for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.,using the in vivo cellular machinery of the host cell rather than invitro manipulations. However, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e., through the expression of a recombinant nucleic acidas depicted above.

The term “variant” as used herein refers to substantially similarsequences. Generally, polypeptide sequence variants possess qualitativebiological activity in common. Further, these polypeptide sequencevariants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity. Also included within themeaning of the term “variant” are homologues of polypeptides of theinvention. A homologue is typically a polypeptide from a differentspecies but sharing substantially the same biological function oractivity as the corresponding polypeptide disclosed herein.

Further, the term “variant” also includes analogues of the polypeptidesof the invention, wherein the term “analogue” means a polypeptide whichis a derivative of a polypeptide of the invention, which derivativecomprises addition, deletion, substitution of one or more amino acids,such that the polypeptide retains substantially the same function. Theterm “conservative amino acid substitution” refers to a substitution orreplacement of one amino acid for another amino acid with similarproperties within a polypeptide chain (primary sequence of a protein).

As for polypeptides discussed above, the term “variant” as used hereinrefers to substantially similar sequences. Generally, polynucleotidesequence variants encode polypeptides which possess qualitativebiological activity in common. Further, these polynucleotide sequencevariants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity. Also included within themeaning of the term “variant” are homologues of polynucleotides of theinvention. A homologue is typically a polynucleotide from a differentspecies but sharing substantially the same activity.

The term “invariant” refers to one or more amino acids orpolynucleotides that do not substantially vary, for example, in relativeposition or charge, in, for example, a class, sub-class, group or familyof amino acid, polypeptide or polynucleotide sequences.

The terms “identical” or percent “identity” in the context of two ormore polypeptide (or nucleic acid) sequences, refer to two or moresequences or sub-sequences that are the same or have a specifiedpercentage of amino acid residues (or nucleotides) that are the sameover a specified region, when compared and aligned for maximumcorrespondence over a comparison window or designated region, asmeasured using sequence comparison algorithms, or by manual alignmentand visual inspection, such techniques being well known to the personskilled in the art.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “similarity”, “sequence similarity”, “sequenceidentity”, “percentage of sequence similarity”, “percentage of sequenceidentity”, “substantially similar” and “substantial identity”. A“reference sequence” is at least 12 but frequently 15 to 18 and often atleast 25 or above, such as 30 monomer units, inclusive of nucleotidesand amino acid residues, in length. Because two polynucleotides may eachcomprise (1) a sequence (i.e. only a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of typically 12 contiguous residues that is comparedto a reference sequence. The comparison window may comprise additions ordeletions (i.e. gaps) of about 20% or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by computerizedimplementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Drive Madison, Wis., USA) or by inspection and thebest alignment (i.e. resulting in the highest percentage homology overthe comparison window) generated by any of the various methods selected.Reference also may be made to the BLAST family of programs as forexample disclosed by Altschul et al. (Nucl. Acids, Res. 25: 3389, 1997).A detailed discussion of sequence analysis can be found in Unit 19.3 ofAusubel et al. (In: Current Protocols in Molecular Biology, John Wiley &Sons Inc. 1994-1998).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp,Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present disclosure, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

The nucleic acid molecules taught herein are also capable of hybridizingto other genetic molecules including the nucleic acid molecule encodingthe heterogeneous defensin.

Stringency conditions can be defined by, for example, the concentrationsof salt or formamide in the pre-hybridization and hybridizationsolutions, or by the hybridization temperature, and are well known inthe art. For example, stringency can be increased by reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature, altering the time ofhybridization, as described in detail, below. In alternative aspects,nucleic acids of the present disclosure are defined by their ability tohybridize under various stringency conditions (e.g., high, medium, andlow).

Reference herein to a “low stringency” includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as “mediumstringency”, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or “high stringency”,which includes and encompasses from at least about 31% v/v to at leastabout 50% v/v formamide and from at least about 0.01 M to at least about0.15 M salt for hybridization, and at least about 0.01 M to at leastabout 0.15 M salt for washing conditions. In general, washing is carriedout T_(m)=69.3+0.41 (G+C) % (Marmur and Doty, J Mol Biol 5:109-118,1962). However, the Tm of a duplex nucleic acid molecule decreases by 1°C. with every increase of 1% in the number of mismatch base pairs(Bonner and Laskey, Eur J Biochem 46:83-88, 1974). Formamide is optionalin these hybridization conditions. Accordingly, particularly preferredlevels of stringency are defined as follows: low stringency is 6×SSCbuffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSCbuffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.;high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of atleast 65° C.

As used herein the term “treatment”, refers to any and all uses whichremedy a disease state or symptoms, prevent the establishment ofdisease, or otherwise prevent, hinder, retard, ameliorate or reverse theprogression of disease or other undesirable symptoms in any waywhatsoever.

The terms “Class I defensin” includes reference to defensins fromvarious organisms such as mammals, plants and insects. Class I plantdefensins sequences may be identified by the presence of an endoplasmicreticulum (ER) signal sequence followed by a mature defensin domain asillustrated in FIG. 10. Examples of Class I defensins as used hereininclude, but are not limited to; NaD2 (Acc No. AF509566); g1-H (Acc No.P20230); Psd1 (Acc No. P81929); Ms-Def1 (Acc No. AAV85437); Dm-AMP1 (AccNo. AAB34972); R5-AFP2 (Acc No. AAA69540) or g-zeathionin 2 (Acc No.ABG78829) see FIG. 11 and FIGS. 13 to 15 for exemplary Class I defensinsequences, origins and Accession numbers.

The terms “Class II defensins” “Class II plant defensins” and “Class IISolanaceous plant defensins” as used herein refer to defensins that areproduced as larger precursors with C-terminal pro-domains orpro-peptides (CTPPs) of about 33 amino acids. Most of the Class IIdefensins identified to date have been found in Solanaceous plantspecies such as Nicotiana spp., Petunia spp., Capsicum spp. Sequencealignments of exemplary Class II Solanaceous plant defensins of theinvention are provided in FIGS. 11 to 15. A Class II Solanaceousdefensin can be generally distinguished from other defensins by arelatively conserved C-terminal domain as shown in FIG. 10. Examples ofClass II Solanaceous defensins as used herein include, but are notlimited to; NaD1 (SEQ ID NO: 22; NCBI database accession no. AF509566),NsD1 (SEQ ID NO 109 and 110), NsD2 (SEQ ID NO: 111 and 112), NoD173 (SEQID NO: 113 and 114), PhD1A (Sol Genomics Network database accession no.SGN-U207537), TPP3 (NCBI database accession no. SLU20591), FST (NCBIdatabase accession no. Z11748), NatD1 (NCBI database accession no.AY456268), NeThio1 (NCBI database accession no. AB005265), NeThio2 (NCBIdatabase accession no. AB005266), NpThio1 (NCBI database accession no.AB005250), CcD1 (NCBI database accession no. AF128239), PhD1 (NCBIdatabase accession no. AF507975), PhD2 (NCBI database accession no.AF507976), any defensin with an amino acid or nucleic acid sequencecorresponding to any of the sequences set forth under NCBI databaseaccession numbers EU367112, EU560901, AF112869 or AF112443, or anydefensin with an amino acid or nucleic acid sequence corresponding toany of the sequences set forth under Sol Genomics Network databaseaccession numbers SGN-U448338, SGN-U449253, SGN-U448480, SGN-U447308,SGN-U578020, SGN-U577258, SGN-U286650, SGN-U268098, SGN-U268098,SGN-U198967, SGN-U196048, SGN-U198968 or SGN-U198966.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g. in cell biology, chemistry, molecular biology and cellculture). Standard techniques used for molecular and biochemical methodscan be found in Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4thEd, John Wiley & Sons, Inc.—and the full version entitled CurrentProtocols in Molecular Biology).

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Throughout this specification, reference to numerical values, unlessstated otherwise, is to be taken as meaning “about” that numericalvalue. The term “about” is used to indicate that a value includes theinherent variation of error for the device and the method being employedto determine the value, or the variation that exists among the studysubjects.

The reference to any prior art in this specification is not, and shouldnot be taken as an acknowledgement or any form of suggestion that priorart forms part of the common general knowledge of the person skilled inthe art.

The entire content of all publications, patents, patent applications andother material recited in this specification is incorporated herein byreference.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is an exemplary amino acid sequence of a heterogeneousdefensin.

SEQ ID NOS: 2-21 and 89-108 are exemplary nucleotide sequences andcorresponding amino acid sequences for the second polynucleotidesequence of the heterogeneous defensin.

SEQ ID NO: 22 is an exemplary full length amino acid sequence for theplant defensin NaD1.

SEQ ID NO: 23 is an exemplary full length amino acid sequence for theplant defensin Dm-AMP 1 and SEQ ID NO:24 is the corresponding nucleotidesequence.

SEQ ID NO: 25 is an exemplary full length amino acid sequence for theplant defensin g1-H and SEQ ID NO:26 is the corresponding nucleotidesequence.

SEQ ID NO: 27 is an exemplary full length amino acid sequence for theplant defensin NaD2 and SEQ ID NO:28 is the corresponding nucleotidesequence.

SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ IDNO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63: SEQ ID NO: 65,SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ IDNO: 85, SEQ ID NO: 87 are exemplary full length amino acid sequences ofheterogeneous defensin.

SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ IDNO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID 76,SEQ ID NO: 78, SEQ ID 80, SEQ ID NO: 82, SEQ ID 84, SEQ ID NO: 86 andSEQ ID 88 are the corresponding nucleic acid sequences of the exemplaryfull length amino acid sequences of heterogeneous defensin.

SEQ ID NO: 109 is an exemplary full length amino acid sequence for theplant defensin NsD1, with SEQ ID NO: 110 being the corresponding nucleicacid sequence.

SEQ ID NO: 111 is an exemplary full length amino acid sequence for theplant defensin NsD2, with SEQ ID NO: 112 being the corresponding nucleicacid sequence.

SEQ ID NO:113 is an exemplary full length amino acid sequence for theplant defensin NoD173 with SEQ ID NO: 114 being the correspondingnucleic acid sequence.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will now be described, by way of example only,with reference to the following figures.

FIG. 1A is a series of micrographs showing the effect of NaD1 on humanHeLa and U937 cells. HeLa cells cultured as adherent monolayers oncoverslips or U937 cells immobilized onto 10% poly-L-lysine-coatedcoverslips, were stained with the membrane dye PKH67 (20 μM) and treatedwith 1001 NaD1 in the presence of 1 μg/ml propidium iodide (PI) for 25minutes. Cells were then imaged by normal light microscopy or confocallaser scanning microscopy (CLSM). Scale bars represent 10 μm.

FIG. 1B is a series of micrographs comparing the kinetics of blebformation and permeabilisation in U937 cells mediated by NaD1. U937cells immobilised onto 10% poly-L-lysine coverslips were treated with200 NaD1 in the presence of both 100 μg/ml 4 kDa FITC-dextran and 1μg/ml PI and cells imaged over a period of 6 minutes. Cells were thenimaged by light microscopy and confocal laser scanning microscopy(CLSM). The arrow indicates the first site of PI entry. Scale barsrepresent 10 μm.

FIG. 2A is a graphical representation of a flow cytometry dot plotshowing BODIPY-NaD1 binding to viable and NaD1-permeabilised U937 cells.U937 cells at 10⁶ ml⁻¹ in RPMI medium containing 0.1% BSA were treatedwith 10 μM NaD1 or BODIPY-NaD1 at 37° C. for 30 min prior to addition of7AAD (1 μg ml⁻¹) and flow cytometry analysis. The percentage of cells ineach population (boxed) is indicated.

FIG. 2B is a series of micrographs examining the subcellularlocalisation of BODIPY-NaD1 in U937 and PC3 cells. U937 cellsimmobilised onto 10% poly-L-lysine coverslips or PC3 cells cultured asadherent monolayers on coverslips, were treated with 1001 BODIPY-NaD1 inthe presence of 1 μg/ml PI for 12 minutes and cells imaged by CLSM.Scale bars represent 10 μm.

FIG. 2C is a series of micrographs showing the effect of NaD1 onGFP-only transfected Hela cells. HeLa cells were cultured as adherentmonolayers on coverslips and transfected with pEGFP-N1. Cells were thentreated with 10 μM NaD1 in the presence of 1 μg/ml PI and imaged bynormal light microscopy and CSLM over a period of 6 minutes. The arrowindicates the first site of PI entry. Scale bars represent 10 μm.

FIG. 2D is a series of micrographs showing the effect of NaD1 onGFP-PH(PLCδ) transfected Hela cells. HeLa cells were cultured asadherent monolayers on coverslips and transfected withpEGFP-GFP-PH(PLCδ). Cells were then treated with 10 μM NaD1 in thepresence of 1 μg/ml PI and imaged by normal light microscopy and CSLMover a period of 6 minutes. The arrow indicates the first site of PIentry on a GFP-PH(PLCδ) expressing cell. Scale bars represent 10 μm.

FIG. 3A is a diagrammatic representation showing the superimposition ofthe NaD1 NMR structure (PDB: 1MR4) with the NaD1 monomer crystalstructure.

FIG. 3B is a series of diagrammatic representations of the NaD1:PIP2(phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2) oligomer. Thetop two panels show two orthogonal views of the NaD1:PIP2 oligomercomprising 14 NaD1 monomers (shown as ribbons) and 14 PIP2 molecules(shown as sticks), with the surface of the complex shown in translucentform. The bottom panel shows a surface representation of the NaD114-mer, displaying the extended binding groove on the inside of thearch. For clarity the 14 bound PIP2 molecules were omitted.

FIG. 3C is a series of diagrammatic representations showing themolecular basis of the monomer-monomer and dimer-dimer interactions. Inthe top left panel, the interface of two NaD1 monomers revealing thehydrogen bonding pattern is shown. Key residues involved in interactionsin the monomer-monomer interface are labelled. For clarity bound PIP2molecules are omitted. Main chain-only hydrogen bonds are formed betweenK4 residues from each monomer, main chain:side chain hydrogen bonds arefound between the backbone oxygen of E6 and R1, and the backbone oxygenof C47 and K45 respectively. Salt bridges are found between R1 and E27from each monomer. In the top right panel, four molecules of NaD1forming a dimer of dimers is shown. Monomer I N8 forms a hydrogen bondwith K17 from monomer III. Monomer II E2 and D31 form hydrogen bondswith the backbone nitrogen of monomer III R1, with the nitrogen ofmonomer II R1 forming a hydrogen bond with monomer III D31. A monomerII:monomer IV hydrogen bond is found between K17 and N8. In the bottomleft panel the PIP2 binding site on monomer I is shown. PIP2interactions with monomer I are mediated by K4, H33, K36, I37, L38 andR40 with all three phosphate groups of PIP2. Additional hydrogen bondsare contributed by monomer II R40 and monomer IV′ K36. The bottom rightpanel shows the PIP2 binding site on monomer II. PIP2 interactions withmonomer II are mediated by K4, H33, K36, I37, L38 and R40 with all threephosphate groups of PIP2. Additional hydrogen bonds are contributed bymonomer I R40 and monomer III K36.

FIG. 3D is a schematic diagram illustrating the molecular interactionsof the NaD1:PIP2 complex. The amino acid residues from neighbouring NaD1monomers involved in binding two PIP2 molecules are illustrated as boxesand their interactions with PIP2 indicated with lines.

FIG. 4A is a series of transmission electron microscopy (TEM) images ofthe NaD1-PIP2 oligomer. Shown are images of NaD1 alone (left panel),PIP2 alone (middle panel) and NaD1-PIP2 (right panel). Scale barsrepresent 100 nm.

FIG. 4B is an immunoblot depicting the lipid-mediated oligomerisation ofthe class II defensins NaD1 and TPP3, and the class I defensin NaD2.Each of the defensins at 250 ng/ml were incubated with 0.5 mM PIP2 or PAat room temperature for 30 minutes and samples then treated with thecross-linker bis[sulfosuccinimidyl] suberate (BS³) at 12.5 mM for afurther 30 minutes, Samples were reduced and denatured, separated bySDS-PAGE, and examined by immunoblotting with an anti-NaD1 (NaD1 andTPP3) or anti-NaD2 (NaD2) antibody. Molecular weight markers areindicated.

FIG. 5A is a diagram depicting the amino acid sequence alignment ofrecombinant NaD1 (rNaD1) and the two NaD1 loop swap forms D2L4A andD2L5. D2L4A and D2L5 differ from rNaD1 in that their loop 4 and loop 5regions have been replaced with the equivalent regions of the class Idefensins NaD2, respectively.

FIG. 5B is a graphical representation showing the effect of rNaD1,rNaD1(D2L4A), or rNaD1(D2L5) on the permeabilisation of U937 cells.Cells were incubated with 10 μM of each defensin for 30 minutes at 37°C. upon which 1 μg/ml PI propidium iodide (PI) was added. The number ofcells that stained positively for PI was determined by flow cytometry.

FIG. 5C is an immunoblot depicting the lipid binding profile of rNaD1,rNaD1(D2L4A), or rNaD1(D2L5). Echelon™ membrane lipid strips were probedwith the defensins and binding detected with a rabbit anti-NaD1 antibodyfollowed by a horseradish peroxidase (HRP) conjugated donkey anti-rabbitIgG antibody.

FIG. 5D is a graphical representation of the densitometricquantification of the lipid binding profile of rNaD1 or rNaD1(D2L4A) orNaD1(D2L5) to Echelon™ membrane lipid strips shown in FIG. 5C. Data isthe mean of three replicate experiments±SEM.

FIG. 6A is a diagram depicting the amino acid sequence of NaD1 and theNaD1 with a loop swap DmAMP1L5, γ1-hordoL5, RsAFP2L5 and VrD1L5. Thefour loop swap NaD1s differ from NaD1 in that their loop 5 regions havebeen replaced with the equivalent regions of the class I defensinsDmAMP1, γ1-hordothionin, RsAFP2 and VrD1.

FIG. 68 is a graphical representation showing the effect of NaD1,DmAMP1L5, γ1-hordoL5, RsAFP2L5 and VrD1L5 on the permeabilisation ofU937 cells. Cells were incubated with 10 μM of each defensin for 30minutes at 37° C. upon which 1 μg/ml PI propidium iodide (PI) was added.The number of cells that stained positively for PI was determined byflow cytometry.

FIG. 6C is an immunoblot depicting the lipid binding profile of NaD1,DmAMP1L5, γ1-hordoL5, RsAFP2L5 and VrD1L5. Echelon™ membrane lipidstrips were probed with the defensins and binding was detected with arabbit anti-NaD1 antibody followed by a horseradish peroxidase (HRP)conjugated donkey anti-rabbit IgG antibody.

FIG. 6D is a graphical representation of the densitometic quantificationof the lipid binding profile of NaD1, NaD1(DmAMP1L5), NaD1(γ1-hordoL5),NaD1(RsAFP2L5) and NaD1(VrD1L5) to Echelon™ membrane lipid strips shownin FIG. 6C. Data is the mean of three replicate experiments±SEM.

FIG. 7 is a diagrammatic representation of the proposed molecularmechanism by which NaD1 induces membrane blebbing/permeabilisation andoligomerisation with PIP2. The proposed four main steps are shown as (i)entry, (ii) dimerisation and competition, (iii) oligomerisation andbleb, and (iv) permeabilisation. The proposed order of assembly ofNaD1:PIP2 oligomer is also shown and can potentially be formed by thesequential recruitment of a NaD1 monomer followed by a PIP2 molecule orthe dimerisation of two single NaD1:PIP2 complex followed by therecruitment of NaD1:PIP2 dimers.

FIG. 8 is a graphical representation showing the effect of rNaD1, K36Eand R40E NaD1 mutants on the permeabilisation of U937 cells. Cells wereincubated with 5 μM of each defensin for 30 minutes at 37° C. upon which1 mg/ml PI propidium iodide (PI) was added. The number of cells thatstained positively for PI was determined by flow cytometry.

FIG. 9 Part A is a ribbon representation of solution structure of NaD1(PDB code 1MR4). The N- and C-termini, all secondary structure elementsand the side-chains of the four disulfide bonds are shown. The Loop 5region is indicated. FIG. 9 Part B is a diagrammatic representation ofthe amino acid sequence of NaD1. The secondary structure elements andthe disulfide bonds (dashed lines) are given above the sequence. Loops(L1-L7), as defined by the amino acids between two neighbouringinvariant cysteine residues, are given below the sequence.

FIG. 10 diagrammatically illustrates the two classes of plant defensins.Part A: Class I: All plant defensins are produced with an ER signalsequence in addition to the mature defensin domain. Part B: Class II: Insome plants, particularly those from the Solanaceae, cDNA clones havebeen isolated that encode plant defensins with an additional C-terminalprodomain. The four strongly conserved disulfide bonds in the defensindomain are illustrated by connecting lines.

FIG. 11 is a representation of exemplary amino acid sequence alignmentsof Class II Solanaceous plant defensins and Class I plant defensins,showing the eight conserved or invariant cysteine residues and otherhighly conserved amino acid residues as shaded regions.

FIG. 12A is a representation of an alignment of exemplary amino acidsequences of Class II Solanaceous plant defensins demonstrating theextremely high level of conservation in the amino acid sequence locatedbetween the fifth and sixth invariant Cysteine residues corresponding tothe loop 5 region (note: CcD1 from Capsicum chinense is identified asCc-gth in this figure).

FIG. 12B is the same representation as shown in FIG. 12A withoutshading.

FIG. 13 is a representation of an alignment of exemplary amino acidsequences of Class I defensins aligned against the Class II Solanaceousdefensin NaD1. It demonstrates the variability amongst Class I defensinsof the amino acid sequence located between the fifth and sixth invariantcysteine residues corresponding to the loop 5 region.

FIG. 14A is a representation of an alignment of exemplary amino acids ofClass II Solanaceous defensins and Class I defensins based on analignment of the corresponding positions of the eight invariant Cysteine(Cys) residues demonstrating that certain amino acid residues outside ofthe loop 5 region are also conserved. The eight conserved or invariantcysteine residues and other conserved amino acid residues are shown asshaded regions. For the sake of comparison, the numbering of all theamino acid sequences in the alignment is based on the NaD1 amino acidsequence whereby the first amino acid residue of NaD1 is designated asresidue 1.

FIG. 14B is the same representation as shown in FIG. 14A withoutshading.

FIG. 15A is a representation of an alignment of exemplary amino acids ofClass II Solanaceous defensins and Class I defensins based on analignment of the eight invariant Cysteine residues again demonstratingthat certain amino acid residues outside of the loop 5 region are alsoconserved. For the sake of comparison, the numbering of all the aminoacid sequences in the alignment is based on the NaD1 amino acid sequencewhereby the first amino acid residue of NaD1 is designated as residue 1.

FIG. 15B is the same representation as shown in FIG. 14A withoutshading.

DETAILED DESCRIPTION OF THE INVENTION

As a result of the findings of the inventors as described herein,heterogeneous forms of plant defensins with advantageous qualities andfunctions, including but not limited to functionalisation of defensinsthat were previously inactive or had low activity in relation totreatment of proliferative disease, new or varied plant defensinfunctions, defensins with improved or enhanced cytotoxic potency,membrane permeabilisation activity and/or broader spectrum of activitycan be produced.

The heterogeneous defensins as described herein are proposed to beuseful in the manufacture of animal and human medicaments as well asassociated uses, systems and kits. These findings also provide formethods for the prevention or treatment of proliferative diseases suchas cancer, as well as associated uses, systems, kits.

Through investigations into the mechanism of action of the defensins,the present inventors have surprisingly found that the Class IISolanaceous plant defensin loop 5 region, as exemplified by NaD1, isinvolved in the anti-proliferative activity of Class II Solanaceousdefensins. Without being bound to any mechanism or theory, the inventorshave identified that specific amino acid residues located in the loop 5region of Class II Solanaceous plant defensins interact with cellularlipids, particularly PIP2 (phosphatidylinositol 4,5-bisphosphate orPtdlns(4,5)P2) in cell membranes.

For example, the inventors observed that addition of 10 μM NaD1 to thehuman tumour cell lines HeLa and PC3 resulted in profound plasmamembrane blebbing and cell permeabilsation (FIG. 1A). Significantly, thesite of cell permeabilisation corresponds with the site of plasmamembrane blebbing, demonstrating NaD1 causes membrane blebbing thatresults in weakening of the plasma membrane and cell permeabilisation(FIG. 1B). NaD1 was found to accumulate on the surface of U937 cellsprior to membrane permeabilisation (FIG. 2A) and inside permeabilisedcells at the membrane bleb(s), cytoplasm, nucleolus and possibly atspecific cytoplasmic organelles (FIG. 2B). Without wishing to be boundby a specific mechanism or theory, the inventors observed that thebinding of NaD1 to the phosphoinositide PIP2 is involved in cellpermeabilisation and tumour cell killing. Further, HeLa cellsoverexpressing the PIP2 binding protein PH(PLCδ) tagged with GFP wereprotected from permeabilisation by NaD1, whereas cells overexpressingGFP alone were sensitive to NaD1 (FIGS. 2C and 2D). These datademonstrate that the binding of PIP2 by NaD1 initiates membrane blebbingand cell permeabilisation and represents a novel mechanism by whichtumour cells can be killed. The understanding of the mechanism of actionof Class II Solanaceous plant defensins, such as NaD1, providesimportant information to assist in the design of novel defensins withenhanced or transformed functions for therapeutic use in the preventionor treatment of proliferative diseases.

The structural determination of the NaD1-PIP2 complex by crystallizationand X-ray diffraction was used to define the precise molecular basis ofhow NaD1 interacts with PIP2 (FIG. 3A-3C). The region of NaD1 that isinvolved for the binding of PIP2 was identified as loop 5, amino acidsSer³⁵-Arg⁴⁰ (FIG. 9). The binding of PIP2 by NaD1 also mediates theformation of an oligomeric structure (FIGS. 4A and 4B). Oligomerformation by NaD1 is lipid-dependent, with the binding of only PIP2 butnot PA mediating the efficient formation of oligomers. Theoligomerisation by PIP2 was also conserved in another class II defensin,TPP3. In contrast, the Class I defensin NaD2 forms oligomers with PA butnot PIP2 (FIG. 4B). Without wishing to be bound by theory, as Class IISolanaceous plant defensins but not Class I defensins are able to killtumour cells, these data suggest that the specificity of lipid-mediatedoligomerisation of defensins is involved in tumour cell killing (FIG.7).

The inventors further observed that the amino acid sequences of a rangeof Class II Solanaceous plant defensins, as shown in FIGS. 11 and 12Aand 12B, are very highly conserved in the loop 5 region flanked by thefifth and sixth invariant cysteines. However, in contrast, there is verylittle consensus across the Class I defensin group as a whole in theloop 5 region. There is also very little, to no consensus between theloop 5 region sequence in the Class II Solanaceous defensins and theloop 5 sequence region in the Class I defensins (see FIGS. 11 to 15).

Without being bound to any specific mechanism or pathway, the inventorshave found that the Class II Solanaceous plant defensin loop 5 region,as defined by being flanked by the fifth and sixth (invariant) cysteine(Cys) amino acid residues of a Class II Solanaceous plant defensin aminoacid sequence, is involved in cytoxicity by replacing of Ser³⁵-Arg⁴⁰ ofNaD1 with the equivalent region of the class I defensins NaD2, DmAMP1,γ1-hordothionin, RsAFP2 or VrD1, and observing a loss in cytotoxicactivity (FIGS. 5A-C and 6A-C). In addition, the replacement of twodifferent residues in the loop 5 region lysine-(K)36 and arginine-(R)40with glutamic acid (E) also resulted greatly reduced cellpermeabilisation (FIG. 8), further demonstrating the involvement of theloop 5 region of Class II plant defensins in tumour cell killing andsuggesting that a Class I defensin could be transformed into a defensinwith cytotoxic activity by replacing its loop 5 region with a loop 5region (for example Ser³⁵-Arg⁴⁰ of NaD1) from a Class II Solanaceousdefensin.

In further studies, the present inventors have identified additionalamino acid residues outside of the Class II Solanaceous defensin loop 5region that are important for the cytoxic activity of the defensin andwhich are involved in, for example any one or more of the formation ofH-bonds and/or salt bridges, protein-PIP2 interactions and/or PI uptake,plasma membrane blebbing and/or cell permeabilisation. Specifically,based on numbering relative to the continuous NaD1 amino acid sequence(SEQ ID NO. 22), the inventors have observed that, in addition to theloop 5 region, any one, or more of the following additional amino acidresidues outside of the Class II Solanaceous defensin loop 5 areimportant for cytoxic activity of Class II Solanaceous defensins:Lysine-(K)4, Lysine-(K)28, Aspartic Acid (D)31, Histidine-(H)33 andLysine (K)45. FIGS. 14 and 15 illustrate the position of theseadditional amino acids in NaD1 relative to other exemplary Class II andClass I plant defensins. Without wishing to be bound by theory, based onthis observation, the inventors have found that, in addition to the loop5 region as mentioned above, the presence of one or more of thefollowing amino acids, at the positions noted, is important forcytotoxic activity:

a K (Lysine) at or around +1 amino acid residue relative to the firstinvariant Cysteine;

a K (Lysine) at or around +5 amino acid residues relative to the fourthinvariant Cysteine;

a D (Aspartic Acid) at or around −3 amino acids residues relative to thefifth invariant Cysteine;

a H (Histidine) at or around −1 amino acid residue relative to the fifthinvariant Cysteine; and/or

a K (Lysine) at or around +2 amino acid residues relative to the seventhinvariant Cystine.

As would be clear to the person skilled in the art, the nomenclatureused herein to describe the invariant Cysteines as, for example, “thefirst invariant Cysteine”, “the second invariant Cysteine”, “the fourthinvariant Cysteine”, etc, refers to the first, second or fourthoccurrence, respectively, of a Cysteine in the relevant sequence whenviewed from left to right as presented herein. The person skilled in theart would therefore understand that reference to a K (Lysine) at oraround +1 amino acid residue relative to the first invariant Cysteinerefers to a K occurring at 1 position to the right of the firstinvariant Cysteine, as presented herein, and similarly, reference to a D(Aspartic Acid) at or around −3 amino acids residues relative to thefifth invariant Cysteine refers to a D occurring at 3 positions to theleft of the fifth invariant Cysteine, as presented herein.

As a result of these studies into plant defensins, a new family ofmutant forms of plant defensins may be produced with advantageousqualities and functions, including but not limited to functionalisationof defensins that were previously inactive or had low activity inrelation to treatment of proliferative diseases, variation of, oraddition of new, plant defensin effector functions, and development ofdefensins with higher cytotoxic potency, by, for example, alteration,substitution or modification of amino acids inside and, optionally;outside of the loop 5 region of a Class I defensin so as to produce anovel heterogeneous defensin, with, for example, the added ability tointeract with phospholipids such as PIP2.

Heterogeneous Plant Defensins

The present invention provides a heterogeneous plant defensin, whereinthe plant defensin comprises a first polypeptide sequence and a secondpolypeptide sequence, wherein the second polypeptide sequence is derivedfrom a plant defensin other than the plant defensin from which the firstpolypeptide sequence is derived.

In some embodiments, the heterogeneous plant defensin comprises a thirdpolypeptide sequence.

In some embodiments, the heterogeneous plant defensin comprises thesequence:

(SEQ ID NO: 1)X_(A)-C1-X_(B)-C2-X_(C)-C3-X_(D)-C4-X_(E)-C5-X_(F)-C6-X_(G)-C7-X_(H)-C8-X_(I);wherein C1, C2, C3, C4, C5, C6, C7 and C8 is cysteine, said cysteinebeing an invariant cysteine,X_(A), X_(B), X_(C), X_(D), X_(E), X_(F), X_(G), X_(H), X_(I) is anynaturally occurring amino acid and;X_(A) is 1 to 7 amino acids in length;X_(B) is 9, 10 or 11 amino acids in length;X_(C) is 3 to 8 amino acids in length;X_(D) is 3 amino acids in length;X_(E) is 9 to 13 amino acids in length;X_(F) is 4, 5, 6, 7 or 8 amino acids in length;X_(G) is 1 amino acid in length;X_(H) is 1 to 4 amino acids in length; andX_(I) is 0 or 1 amino acid in length.

In some embodiments, the second polypeptide sequence is positionedbetween the fourth and eighth invariant cysteine amino acid residuescorresponding to the sequence as set forth in SEQ ID NO:1

In some embodiments, the second polypeptide is positioned between thefifth and sixth invariant cysteine residues corresponding to thesequence as set forth in SEQ ID NO:1.

In preferred embodiments the first polypeptide sequence is derived froma Class I plant defensin.

In preferred embodiments the second polypeptide sequence is derived froma Class II Solanaceous plant defensin.

In certain embodiments, the first polypeptide sequence is derived from aClass I plant defensin and the second polypeptide sequence is derivedfrom a Class II Solanaceous plant defensin.

In some embodiments the third polypeptide sequence is derived from aClass I plant defensin.

In some embodiments, the second polypeptide sequence comprises a loop 5region derived from a Class II plant defensin, or a fragment or variantthereof. The loop 5 region may begin with a serine.

In some embodiments, the loop 5 region derived from the Class IISolanaceous plant defensin comprises the entire loop 5 region of a ClassII Solanaceous plant defensin, said loop 5 defined as by being flankedby the fifth and sixth invariant cysteine (Cys) amino acid residues of aClass II Solanaceous plant defensin amino acid sequence.

In some embodiments, the second polypeptide sequence comprises afragment or modified form of the loop 5 region of a Class II Solanaceousplant defensin.

In certain embodiments, loop 5 region derived from the Class IISolanaceous plant defensin comprises an amino acid sequence beginning atthe fifth invariant cysteine amino acid residue, or end of the secondβ-strand, and ending at the N-terminal side of the sixth invariantcysteine amino acid residue of a Class II Solanaceous plant defensinamino acid sequence. The loop 5 region is referred to as “L5” in FIG. 9Part B. Exemplary Class II Solanaceous plant defensin loop 5 regions andamino acid sequences are shown in FIGS. 11, 12A and 12B.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serineor arginine, X₂ is lysine, arginine or histidine, X₃ and X₄ are each ahydrophobic amino acid, X₅ is arginine, lysine or histidine and X₆ isarginine, lysine, histidine, asparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serineor arginine, X₂ is lysine, X₃ is isoleucine, leucine or valine, X₄ isleucine or glutamine, X₅ is arginine and X₆ is arginine, lysine orasparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine,arginine or histidine, X₃ and X₄ are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆ is arginine, lysine, histidineor asparagine.

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, X₃is isoleucine, leucine or valine, X₄ is leucine or glutamine, X₅ isarginine and X₆ is arginine, lysine or asparagine.

In some embodiments, the second polypeptide corresponds to an amino acidsequence comprising (i) SKILRR (SEQ ID NO: 2), (ii) SKLLRR (SEQ ID NO:4), (iii) SKILRK (SEQ ID NO: 6), (iv) SKVLRR (SEQ ID NO: 8), (v) SKVLRK(SEQ ID NO: 10), (vi) SKLQRK (SEQ ID NO: 12), (vii) SKLLRN (SEQ ID NO:14), (viii) SKLLRK (SEQ ID NO: 16), (ix) SKIQRN (SEQ ID NO: 18), (x)RKLQRK (SEQ ID NO: 20) or (xi) KILRR (SEQ ID NO: 89), (xii) KLLRR (SEQID NO: 91), (xiii) KILRK (SEQ ID NO: 93), (xiv) KVLRR (SEQ ID NO: 95),(xv) KVLRK (SEQ ID NO: 97), (xvi) KLQRK (SEQ ID NO: 99), (xvii) KLLRN(SEQ ID NO: 101), (xviii) KLLRK (SEQ ID NO: 103), (xix) KIQRN (SEQ IDNO: 105) or (xx) KLQRK (SEQ ID NO: 107).

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising S-K-I-L-R-R (SEQ ID NO: 2).

In some embodiments, the second polypeptide sequence corresponds to anamino acid sequence comprising K-I-L-R-R (SEQ ID NO: 89).

In some embodiments, the first and the third polypeptide sequences areboth derived from the same plant defensin.

In some embodiments the heterogeneous defensin may display enhancedand/or broader spectrum anti-proliferative disease activity, and/orcytotoxic activity, and/or cell membrane permeabilisation, and/orenhanced PIP2 binding activity relative to a defensin prior tomodification or relative to the defensin from which the firstpolypeptide is derived.

In certain embodiments the heterogeneous defensin binds to aphospholipid, preferably PIP2.

In some embodiments the second polypeptide sequence, or part thereof,binds to PIP2 in a cell membrane.

In preferred embodiments, the heterogeneous plant defensin binds PIP2 ina cell membrane.

In certain embodiments, the heterogeneous plant defensin has cellmembrane permeablisation activity.

In specific embodiments, the cell membrane is a tumour cell membrane.

In some embodiments, first polypeptide sequence and optionally the thirdpolypeptide sequence contain one or more amino acid or nucleotidesubstitutions, deletions or modifications, wherein the substitution,deletion or modification does not naturally occur at the substituted,deleted or modified position in the plant defensin from which the firstpolypeptide sequence and optionally the third polypeptide sequence isderived.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region, said loop 5 region beingpositioned between the fifth and sixth invariant cysteine residues ofthe heterogeneous plant defensin amino acid sequence, and wherein thesecond polypeptide sequence corresponds to an amino acid sequencecomprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serine or arginine, X₂ islysine, arginine or histidine, X₃ and X₄ are each a hydrophobic aminoacid, X₅ is arginine, lysine or histidine and X₆ is arginine, lysine,histidine or asparagine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region, said loop 5 region beingpositioned between the fifth and sixth invariant cysteine residues ofthe heterogeneous plant defensin amino acid sequence, and wherein thesecond polypeptide sequence corresponds to an amino acid sequencecomprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serine or arginine, X₂ islysine, X₃ is isoleucine, leucine or valine, X₄ is leucine or glutamine,X₅ is arginine or lysine and X₆ is arginine or lysine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least invariant eight cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region of the said loop 5 regionbeing positioned between the fifth and sixth cysteine residues of theheterogeneous plant defensin amino acid sequence, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serine or arginine, X₂ is lysine, X₃ isisoleucine, X₄ is leucine, X₅ is arginine and X₆ is arginine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region positioned between thefifth and sixth invariant cysteine residues, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, arginine or histidine, X₃ and X₄are each a hydrophobic amino acid, X₅ is arginine, lysine or histidineand X₆ is arginine, lysine, histidine or asparagine.

In particular embodiments, there is provided a heterogeneous, plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region positioned between thefifth and sixth invariant cysteine residues, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, X₃ is isoleucine, leucine orvaline, X₄ is leucine or glutamine, X₅ is arginine or lysine and X₆ isarginine or lysine.

In particular embodiments, there is provided a heterogeneous plantdefensin, wherein said defensin comprises a first polypeptide sequenceand a second polypeptide sequence and at least eight invariant cysteineamino acid residues, wherein the first polypeptide sequence is derivedfrom a Class I plant defensin and the second polypeptide is derived froma Class II Solanaceous plant defensin, and wherein the secondpolypeptide sequence comprises a loop 5 region positioned between thefifth and sixth invariant cysteine residues, and wherein the secondpolypeptide sequence corresponds to an amino acid sequence comprisingX₂-X₃-X₄-X₅-X₆, wherein X₂ is lysine, X₃ is isoleucine, X₄ is leucine,X₅ is arginine and X₆ is arginine.

In some embodiments, the heterogeneous plant defensin is produced byreplacing the amino acids substantially comprising a loop 5 regionpositioned between the fifth and sixth invariant cysteine residues of aClass I defensin with an amino acid sequence substantially comprising aloop 5 region positioned between the fifth and sixth invariant cysteineresidues of a Class II Solanaceous plant defensin.

In particular embodiments, the heterogeneous plant defensin is selectedfrom the following SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ IDNO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 SEQ ID NO:30, SEQ ID NO: 32, SEQID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO: 60, SEQ IDNO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88.

In certain embodiments, the heterogeneous plant defensin has enhancedand/or broader spectrum anti-proliferative disease, and/or enhancedand/or broader spectrum cytotoxic activity relative to the defensin fromwhich the first polypeptide is derived,

In some embodiments, the heterogeneous plant defensin further comprisesone or more additional amino acid substitutions, deletions or additions,

In some embodiments, the heterogeneous plant defensin further comprisesone or more amino acid substitutions, deletions or additions in thefirst polypeptide.

In particular embodiments, the heterogeneous plant defensin furthercomprises one or more amino acids selected from the group consisting of:a K (Lysine) at or around +1 amino acid residue relative to the firstinvariant Cysteine; a K (Lysine) at or around +5 amino acid residuesrelative to the fourth invariant Cysteine; a D (Aspartic Acid) at oraround −3 amino acids residues relative to the fifth invariant Cysteine;a H (Histidine) at or around −1 amino acid residue relative to the fifthinvariant Cysteine; and/or a K (Lysine) at or around +2 amino acidresidues relative to the seventh invariant Cystine; and/or conservativesubstitutions or functional and/or structural equivalents thereof.

In yet further embodiments, the heterogeneous plant defensin comprises afirst polypeptide derived from a Class I defensin from Aesculushippocastanum, Arabidopsis halleri, Arachis diogoi, Brassica campestris,Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapasubsp., Beta vulgaris, Bupleurum kaoi, Cajanus cajan, Capsicum annuum,Capsicum chinense, Cassia fistula, Cicer arietinum, Clitoria ternatea,Dahlia merckii, Echinochloa crus-galli, Elaeis guineensis, Ginkgobiloba, Glycine max, Hardenbergia violacea, Helianthus annuus, Heucherasanguinea, Hordeum vulgare, Jatropha curcas, Lepidium meyenii, Medicagosativa, Medicago truncatula, Nicotiana alata, Nicotiana tabacum, Oryzasativa Japonica, Pachyrhizus erosus, Petunia integrifolia, Petuniahybrida, Picea abies, Pinus sylvestris, Pisum sativum, Plantago major,Prunus persica, Pyrus pyrifolia, Raphanus sativus, Saccharumofficinarum, Sinapis alba, Solanum lycopersicum, Solanum tuberosum,Sorghum bicolor, Spinacia oleracea, Tephrosia villosa, Trigonellafoenum-gr, Triticum aestivum, Triticum kiharae, Triticum monococcum,Triticum turgidum, Vicia faba, Vigna radiate, Vigna unguiculata, Wasabijaponica, Zea mays, Zea mays subsp. Mays.

In yet further embodiments, the heterogeneous plant defensin comprises afirst polypeptide derived from a Class I defensin from Nicotiana alata,Nicotiana suaveolens, Hordeum vulgare, Pisum sativum, Medicago saliva,Dahlia merckii, Raphanus sativus, Zea mays.

In particular embodiments, the first polypeptide is derived from a ClassI defensin defensin from the group comprising; NaD2 (SEQ ID NO: 27),NsD3, TGAS118 (Acc AJ133601), P322 (Acc ACJ26760), PPT (Acc AAA64740),SE60 (Acc Q0752), 10 kDa (Acc P18646), Cp-thionin, (Acc P83399), VrD1(vrCRP) (Acc AAR08912), Psd1 (P81929), MsDef1 (alfAFP)(Acc AF319468),R5-AFP2 (AccP30230), Ah-AMP1 (Acc AAB34970), Hs-AFP1 (Acc POC8Y5),Dm-AMP1 (Acc AAB34972), Ct-AMP1 (AAB34971), SPI1 (Acc CAA62761), M2A(Acc P30232), SD2 (AAF72042, γ1-H (g1-H) (Acc P20230), γ1-P (AccP20158), Tad1 (Acc BAC10287), Slα2 (Acc P21923), Slα3 (Acc P2192), γ1-Z(Acc P81008), γ2-Z (Acc P81009), Fabatin-1 (Acc P81456), So-D2 (AccP81571) or WT1 (Acc AB012871) (See FIG. 11).

In particular embodiments, the first polypeptide is derived from a ClassI defensin defensin from the group comprising; NaD2, Dm-AMP1, g1-H,Psd1, Ms-Deft, R5-AFP2 or g-zeathionin 2 (γ2-Z).

In particular embodiments, the second polypeptide is derived from aClass II Solanaceous defensin from Nicotiana spp., Solanum spp., Petuniaspp., or Capsicum spp.

In particular embodiments, the second polypeptide is derived from aClass II Solanaceous defensin from Nicotiana alata, Nicotianasuaveolens, Nicotiana occidentalis, Petunia hybrida, Solanumlycopersicum or Capsicum chinense.

In preferred embodiments, the second polypeptide is derived from NaD1,NsD1, NsD2, NoD173, TPP3, PhD1, PhD1A, PhD2, TPP3, FST, NeThio1,NeThio2, NpThio1, Na-gth or CcD1.

Polynucleotides

In embodiments where the compositions of the present invention comprisepolypeptides, the present invention also provides nucleic acids encodingsuch polypeptides, or fragments or complements thereof. Such nucleicacids may be naturally occurring or may be synthetic or recombinant.

In some embodiments, the nucleic acids may be operably linked to one ormore promoters.

In particular embodiments the nucleic acids encode a heterogeneous plantdefensin or a functional fragment thereof.

In more particular embodiments, the heterogeneous plant defensincomprises the amino acid sequence set forth as any one of SEQ ID NO:29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ IDNO: 87.

In particular embodiments, the heterogeneous plant defensin is encodedby the nucleic acid sequence set forth as any one of SEQ ID NO:30, SEQID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ, IDNO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO:88

In some embodiments, the heterogeneous plant defensin comprises a serineamino acid positioned adjacent to the fifth invariant cysteine aminoacid corresponding to the sequence set forth as SEQ ID NO:1, wherein theserine amino acid is positioned to the C-terminal side of the fifthinvariant cysteine.

In still other embodiments, the heterogeneous plant defensin comprisesan amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%_(;) 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%,63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%,49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%,35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%,21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% identical to the amino acid sequence set forthas SEQ ID NOs: SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ IDNO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or a fragment thereof.

Vectors, Host Cells and Expression Products

The present invention also provides vectors comprising the nucleic acidsas set forth herein. The vector may be a plasmid vector, a viral vector,or any other suitable vehicle adapted for the insertion of foreignsequences, its introduction into cells and the expression of theintroduced sequences. The vector may be a eukaryotic expression vectorand may include expression control and processing sequences such as apromoter, an enhancer, ribosome binding sites, polyadenylation signalsand transcription termination sequences. In preferred embodiments, thevector comprises one or more nucleic acids operably encoding any one ormore of the plant defensins set forth herein.

The present invention further provides host cells comprising the vectorsas set forth herein. Typically, a host cell is transformed, transfectedor transduced with a vector, for example, by using electroporationfollowed by subsequent selection of transformed, transfected ortransduced cells on selective media. The resulting heterologous nucleicacid sequences in the form of vectors and nucleic acids inserted thereinmay be maintained extrachromosomally or may be introduced into the hostcell genome by homologous recombination. Methods for such cellulartransformation, transfection or transduction are well known to those ofskill in the art. Guidance may be obtained, for example, from standardtexts such as Sambrook of al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989 and Ausubel of al., Current Protocols inMolecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992.

The present invention moreover provides expression products of the hostcells as set forth herein.

The present invention also provides a vector comprising a nucleic acidencoding the heterogeneous plant defensin or a functional fragmentthereof and a host cell comprising such a vector. In further aspects ofthe invention there is provided a heterogeneous plant defensin producedby the host cell

In some embodiments, the expression product may be polypeptides thatprevent or treat proliferative diseases. In preferred embodiments, theexpression product is any one or more of the plant defensins disclosedherein.

In other embodiments, the isolated nucleic acid molecule may also be ina vector including an expression or transfer vector suitable for use inmicrobial cells and non-human animal cells.

In some embodiments, the polynucleotides as described herein may beinserted within a coding region expressing another protein to form aheterogeneous defensin fusion protein or may be used to replace a domainof a protein to give that protein, anti-proliferative disease activityor cytotoxicity. The nucleic acid sequence may be placed under thecontrol of a homologous or heterologous promoter which may be aconstitutive or an inducible promoter (stimulated by, for example,presence of a chemical). The transit peptide may be homologous orheterologous to the modified defensin and is chosen to ensure secretionto the desired organelle or to the extracellular space. The transitpeptide may be naturally associated with a particular defensin. Such aDNA construct may be cloned or transformed into a biological systemwhich allows expression of the encoded heterogeneous defensin, an activepart of the defensin or fragment thereof, Suitable biological systemsinclude microorganisms (for example, the Pichia pastoris expressionsystem, Escherichia coli, Pseudomonas, yeast; viruses; bacteriophages;etc) and cultured cells (such as insect cells, mammalian cells). In somecases, the expressed defensin is subsequently extracted and isolated foruse.

Compositions

The present invention also provides pharmaceutical compositions, whereinthe pharmaceutical compositions comprise the heterogeneous plantdefensin, a nucleic acid, a vector, a host cell or an expression productas disclosed herein, together with a pharmaceutically acceptablecarrier, diluent or excipient.

Compositions of the present invention may therefore be administeredtherapeutically. In such applications, compositions may be administeredto a subject already suffering from a condition, in an amount sufficientto cure or at least partially arrest the condition and anycomplications. The quantity of the composition should be sufficient toeffectively treat the patient. Compositions may be prepared according tomethods which are known to those of ordinary skill in the art andaccordingly may include a cosmetically or pharmaceutically acceptablecarrier, excipient or diluent. Methods for preparing administrablecompositions are apparent to those skilled in the art, and are describedin more detail in, for example, Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa., incorporated by referenceherein.

The composition may incorporate any suitable surfactant such as ananionic, cationic or non-ionic surfactant such as sorbitan esters orpolyoxyethylene derivatives thereof. Suspending agents such as naturalgums, cellulose derivatives or inorganic materials such as silicaceoussilicas, and other ingredients such as lanolin, may also be included.

The compositions may also be administered in the form of liposomes.Liposomes may be derived from phospholipids or other lipid substances,and may be formed by mono- or multi-lamellar hydrated liquid crystalsdispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolisable lipid capable of forming liposomes may beused. The compositions in liposome form may contain stabilisers,preservatives and excipients. Preferred lipids include phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsfor producing liposomes are known in the art, and in this regardspecific reference is made to: Prescott, Ed., Methods in Cell Biology,Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., thecontents of which are incorporated herein by reference.

In some embodiments, the composition may be in the form of a tablet,liquid, lotion, cream, gel, paste or emulsion.

In some embodiments, the composition may further comprisepharmaceutically or veterinarily acceptable carriers, diluents orexcipients.

In certain embodiments the composition is in the form of a spray, mist,micro- or nano-particles, aqueous solution, powder, cream, ointment,gel, impregnated bandage, liquid, formulation, paint or other suitabledistribution medium including oral forms of the composition.

In particular embodiments the composition further comprises one or moreadditional anti-proliferative disease or cytotoxic agents.

In certain embodiments, the heterogeneous defensin may have beenchemically synthesized or extracted from microorganisms or plantsgenetically modified to express the heterogeneous defensin.

Dosages

The “therapeutically effective” dose level for any particular patientwill depend upon a variety of factors including the condition beingtreated and the severity of the condition, the activity of the compoundor agent employed, the composition employed, the age, body weight,general health, sex and diet of the patient, age, size, growth phase,species of plant, the time of administration, the route ofadministration, the rate of sequestration of the heterogeneous plantdefensin or composition, the duration of the treatment, and any drugs oragents used in combination or coincidental with the treatment, togetherwith other related factors well known in the art. A physician, clinicianor veterinarian of ordinary skill can readily determine and prescribethe effective amount of the defensin required to prevent or treatapplicable conditions.

In certain embodiments, the treatment would be for the duration of thedisease state. Slow release formulations are also contemplated herein.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and spacing of individual dosages of thecomposition will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and thenature of the particular individual being treated. Also, such optimumconditions can be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as the number of doses of thecomposition given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

In terms of weight, a therapeutically effective dosage of a compositionfor administration to a patient is expected to be in the range of about0.01 mg to about 150 mg per kg body weight per 24 hours; typically,about 0.1 mg to about 150 mg per kg body weight per 24 hours; about 0.1mg to about 100 mg per kg body weight per 24 hours; about 0.5 mg toabout 100 mg per kg body weight per 24 hours; or about 1.0 mg to about100 mg per kg body weight per 24 hours. More typically, an effectivedose range is expected to be in the range of about 5 mg to about 50 mgper kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 5000 mg/m².Generally, an effective dosage is expected to be in the range of about10 to about 5000 mg/m², typically about 10 to about 2500 mg/m², about 25to about 2000 mg/m², about 50 to about 1500 mg/m², about 50 to about1000 mg/m², or about 75 to about 600 mg/m².

In particular embodiments, the composition as described herein can beused as part of a soil or growth medium preparation program.

In some embodiments, the composition is formulated as dragee cores withsuitable coatings. For this purpose, concentrated sugar solutions may beused, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

In particular embodiments, the composition is formulated as push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches and/or lubricants such as talcor magnesium stearate and, optionally, stabilizers. In soft capsules,the active compounds may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

Routes of Administration

The compositions of the present invention can be administered bystandard routes. In general, the compositions may be administered by theparenteral (e.g., intravenous, intraspinal, subcutaneous orintramuscular), oral or topical route.

In other embodiments, the compositions may be administered by otherenteral/enteric routes, such as rectal, sublingual or sublabial, or viathe central nervous system, such as through epidural, intracerebral orintracerebroventricular routes. Other locations for administration mayinclude via epicutaneous, transdermal, intradermal, nasal,intraarterial, intracardiac, intraosseus, intrathecal, intraperitoneal,intravesical, intravitreal, intracavernous, intravaginal or intrauterineroutes.

In preferred embodiments, topical compositions can be used. In preparingthe compositions, usual media may be employed such as, for example,water, glycols, oils, alcohols, preservatives and/or coloring agents.

In other embodiments, the modified defensins herein may be administereddirectly to the skin, hair or fur of an animal including a mammal suchas a human.

When administered by aerosol or spray, the compositions are preparedaccording to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons and/or other solubilizing or dispersingagents known in the art.

Carriers, Excipients and Diluents

Carriers, excipients and diluents must be “acceptable” in terms of beingcompatible with the other ingredients of the composition, and notdeleterious to the recipient thereof. Such carriers, excipients anddiluents may be used for enhancing the integrity and half-life of thecompositions of the present invention. These may also be used to enhanceor protect the biological activities of the compositions of the presentinvention.

Examples of pharmaceutically acceptable carriers or diluents aredemineralised or distilled water; saline solution; vegetable based oilssuch as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil,sesame oils, arachis oil or coconut oil; silicone oils, includingpolysiloxanes, such as methyl polysiloxane, phenyl polysiloxane andmethylphenyl polysolpoxane; volatile silicones; mineral oils such asliquid paraffin, soft paraffin or squalane; cellulose derivatives suchas methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols,for example ethanol or iso-propanol; lower aralkanols; lowerpolyalkylene glycols or lower alkylene glycols, for example polyethyleneglycol, polypropylene glycol, ethylene glycol, propylene glycol,1,3-butylene glycol or glycerin; fatty acid esters such as isopropylpalmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone;agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, thecarrier or carriers will form from 10% to 99.9% by weight of thecompositions.

The compositions of the invention may be in a form suitable foradministration by injection, in the form of a formulation suitable fororal ingestion (such as capsules, tablets, caplets, elixirs, forexample), in the form of an ointment, cream or lotion suitable fortopical administration, in an aerosol form suitable for administrationby inhalation, such as by intranasal inhalation or oral inhalation, in aform suitable for parenteral administration, that is, subcutaneous,intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxicacceptable diluents or carriers can include Ringer's solution, isotonicsaline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Methods for Preventing or Treating Disease

The present invention provides methods for preventing or treating aproliferative disease, wherein the method comprises administering to asubject a therapeutically effective amount of the heterogeneous plantdefensin a nucleic acid, a vector, a host cell, an expression product,or a pharmaceutical composition as disclosed herein, thereby preventingor treating the proliferative disease.

The present invention also provides use of plant defensins, nucleicacids, vectors, host cells and expression products as herein disclosedin the preparation of medicaments for preventing or treating aproliferative disease.

In some embodiments, the proliferative disease may be a cellproliferative disease selected from the group comprising an angiogenicdisease, a metastatic disease, a tumourigenic disease, a neoplasticdisease and cancer.

In some embodiments, the proliferative disease may be cancer.

In other embodiments, the cancer may be selected from the groupcomprising acute lymphoblastic leukemia, acute myeloid leukemia,adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendixcancer, astrocytoma, B-cell lymphoma, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, bowel cancer, brainstem glioma,brain tumour, breast cancer, bronchial adenomas/carcinoids, Burkitt'slymphoma, carcinoid tumour, cerebral astrocytoma/malignant glioma,cervical cancer, childhood cancers, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic myeloproliferative disorders,colon cancer, cutaneous T-cell lymphoma, desmoplastic small round celltumour, endometrial cancer, ependymoma, esophageal cancer, extracranialgerm cell tumour, extragonadal germ cell tumour, extrahepatic bile ductcancer, eye cancer, intraocular melanoma/retinoblastoma, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumour,gastrointestinal stromal tumour (GIST), germ cell tumour, gestationaltrophoblastic tumour, glioma, gastric carcinoid, head and/or neckcancer, heart cancer, hepatocellular (liver) cancer, hypopharyngealcancer, hypothalamic and visual pathway glioma, Kaposi sarcoma, kidneycancer, laryngeal cancer, leukemia (acute lymphoblastic/acutemyeloid/chronic lymphocytic/chronic myelogenous/hairy cell), lip and/ororal cavity cancer, liver cancer, non-small cell lung cancer, small celllung cancer, lymphoma (AIDS-related/Burkitt/cutaneousT-Cell/Hodgkin/non-Hodgkin/primary central nervous system),macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma,metastatic squamous neck cancer, mouth cancer, multiple endocrineneoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosisfungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferativediseases, myelogenous leukemia, myeloid leukemia, myeloproliferativedisorders, nasal cavity and/or paranasal sinus cancer, nasopharyngealcarcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lungcancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignantfibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer,ovarian germ cell tumour, pancreatic cancer, islet cell cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pinealgerminoma, pineoblastoma and/or supratentorial primitive neuroectodermaltumours, pituitary adenoma, plasma cell neoplasia/multiple myeloma,pleuropulmonary blastoma, primary central nervous system lymphoma,prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, Kaposi sarcoma,soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin cancer(non-melanoma), skin cancer (melanoma), skin carcinoma (Merkel cell),small cell lung cancer, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma, squamous neck cancer with metastatic occultprimary, stomach cancer, supratentorial primitive neuroectodermaltumour, T-cell lymphoma, testicular cancer, throat cancer, thymomaand/or thymic carcinoma, thyroid cancer, transitional cancer,trophoblastic tumour, ureter and/or renal pelvis cancer, urethralcancer, uterine endometrial cancer, uterine sarcoma, vaginal cancer,visual pathway and hypothalamic glioma, vulva cancer, Waldenstrommacroglobulinemia or Wilms tumour.

In particular embodiments the cancer is selected from the groupcomprising basal cell carcinoma, bone cancer, bowel cancer, braincancer, breast cancer, cervical cancer, leukemia, liver cancer, lungcancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostatecancer or thyroid cancer.

In preferred embodiments, a heterogeneous plant defensin, a nucleicacid, a vector, a host cell, an expression product, or a pharmaceuticalcomposition as disclosed herein, is used in the preparation of amedicament for preventing or treating a proliferative disease,

In preferred embodiments the heterogeneous plant defensin ashereindescribed is used for preventing or treating a proliferativedisease.

Kits

The present invention provides kits for preventing or treating aproliferative disease, wherein the kits comprise a therapeuticallyeffective amount of a heterologous plant defensin, a nucleic acid, avector, a host cell, an expression product or a pharmaceuticalcomposition as herein disclosed.

The present invention also provides use of the kits disclosed herein forpreventing or treating a proliferative disease, wherein thetherapeutically effective amount of a plant defensin, a nucleic acid, avector, a host cell, an expression product or a pharmaceuticalcomposition as herein disclosed is administered to a subject, therebypreventing or treating the proliferative disease,

Kits of the present invention facilitate the employment of the methodsof the present invention. Typically, kits for carrying out a method ofthe invention contain all the necessary reagents to carry out themethod. For example, in one embodiment, the kit may comprise a plantdefensin, a polypeptide, a polynucleotide, a vector, a host cell, anexpression product or a pharmaceutical composition as herein disclosed.

Typically, the kits described herein will also comprise one or morecontainers. In the context of the present invention, a compartmentalisedkit includes any kit in which compounds or compositions are contained inseparate containers, and may include small glass containers, plasticcontainers or strips of plastic or paper. Such containers may allow theefficient transfer of compounds or compositions from one compartment toanother compartment whilst avoiding cross-contamination of samples, andthe addition of agents or solutions of each container from onecompartment to another in a quantitative fashion.

Typically, a kit of the present invention will also include instructionsfor using the kit components to conduct the appropriate methods.

Methods and kits of the present invention are equally applicable to anyanimal, including humans and other animals, for example includingnon-human primate, equine, bovine, ovine, caprine, leporine, avian,feline and canine species. Accordingly, for application to differentspecies, a single kit of the invention may be applicable, oralternatively different kits, for example comprising compounds orcompositions specific for each individual species, may be required.

Methods and kits of the present invention find application in anycircumstance in which it is desirable to prevent or treat aproliferative disease.

In preferred embodiments, the proliferative disease is cancer.

In particular embodiments, the cancer is selected from the groupcomprising basal cell carcinoma, bone cancer, bowel cancer, braincancer, breast cancer, cervical cancer, leukemia, liver cancer, lungcancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostatecancer or thyroid cancer.

Combination Therapies

Those skilled in the art will appreciate that the polypeptides, nucleicacids, vectors, host cells, expression products and compositionsdisclosed herein may be administered as part of a combination therapyapproach, employing one or more of the polypeptides, nucleic acids,vectors, host cells, expression products and compositions disclosedherein in conjunction with other therapeutic approaches to the methodsdisclosed herein. For such combination therapies, each component of thecombination may be administered at the same time, or sequentially in anyorder, or at different times, so as to provide the desired therapeuticeffect. When administered separately, it may be preferred for thecomponents to be administered by the same route of administration,although it is not necessary for this to be so. Alternatively, thecomponents may be formulated together in a single dosage unit as acombination product. Suitable agents which may be used in combinationwith the compositions of the present invention will be known to those ofordinary skill in the art, and may include, for example,chemotherapeutic agents, radioisotopes and targeted therapies such asantibodies.

Chemotherapeutic agents to be used in combination with the polypeptides,nucleic acids, vectors, host cells, expression products and compositionsdisclosed herein may include alkylating agents such as cisplatin,carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil and ifosfamide, anti-metabolites such as purine orpyramidine, plant alkaloids and terpenoids such as vinca alkaloids(including vincristine, vinblastine, vinorelbine and vindesine), andtaxanes (including paclitaxel and docetaxel), podophyllotoxin,topoisomerase inhibitors such as irinotecan, topotecan, amsacrine,etoposide, etoposide phosphate and teniposide, anti-neoplastics such asdoxorubicin, epirubicin and bleomycin, and tyrosine kinase inhibitors.

Targeted therapies to be used in combination with the polypeptides,nucleic acids, vectors, host cells, expression products and compositionsdisclosed herein may include, for example, imatinib mesylate, dasatinib,nilotinib, trastuzumab, lapatinib, gefitinib, erlotinib, cetuximab,panitumumab, temsirolimus, everolimus, vorinostat, romidepsin,bexarotene, alitretinoin, tretinoin, bortezomib, pralatrexate,bevacizumab, sorafenib, sunitinib, pazopanib, rituximab, alemtuzumab,ofatumuab, tositumomab, 131I-tositumomab, ibritumomab tiuxetan,denileukin diftitox, tamoxifen, toremifene, fulvestrant, anastrozole,exemestane and letrozole.

Other therapies may also be used in combination with the polypeptides,nucleic acids, vectors, host cells, expression products and compositionsdisclosed herein, including, for example, surgical intervention, dietaryregimes and supplements, hypnotherapy, alternative medicines andphysical therapy.

Timing of Therapies

Those skilled in the art will appreciate that the polypeptides,polynucleotides, vectors, host cells, expression products andcompositions disclosed herein may be administered as a single agent oras part of a combination therapy approach to the methods disclosedherein, either at diagnosis or subsequently thereafter, for example, asfollow-up treatment or consolidation therapy as a compliment tocurrently available therapies for such treatments. The polypeptides,polynucleotides, vectors, host cells, expression products andcompositions disclosed herein may also be used as preventative therapiesfor subjects who are genetically or environmentally predisposed todeveloping such diseases.

The person skilled in the art will understand and appreciate thatdifferent features disclosed herein may be combined to form combinationsof features that are within the scope of the present invention.

The present invention will now be further described with reference tothe following examples, which are illustrative only and non-limiting.

Examples Materials and Methods

Purification of Defensins from Solanaceous Flowers

To isolate Class II Solanaceous defensins from their natural source,whole flowers up to the petal coloration stage of flower developmentwere ground to a fine powder and extracted in dilute sulfuric acid aspreviously described previously (Lay et al, 2003a supra). Briefly,flowers (760 g wet weight) were frozen in liquid nitrogen, ground to afine powder in a mortar and pestle, and homogenized in 50 mM sulfuricacid (3 mL per g fresh weight) for 5 min using an Ultra-Turraxhomogenizer. The homogenate was transferred to a beaker and stirred for1 h at 4° C. Cellular debris was removed by filtration through Miracloth(Calbiochem, San Diego, Calif.) and centrifugation (25,000×g, 15 min, 4°C.). The pH was then adjusted to 7.0 by addition of 10 M NaOH and theextract was stirred for 1 h at 4° C. before centrifugation (25,000×g, 15min, 4° C.) to remove precipitated proteins. The supernatant was appliedto an SP-Sepharose™ Fast Flow (GE Healthcare Bio-Sciences) column(2.5×2.5 cm) pre-equilibrated with 10 mM sodium phosphate buffer.Unbound proteins were removed by washing with 20 column volumes of 10 mMsodium phosphate buffer (pH 6.0) and bound proteins were eluted in 3×10mL fractions with 10 mM sodium phosphate buffer (pH 6.0) containing 500mM NaCl.

Fractions from the SP Sepharose column were subjected to reverse-phasehigh performance liquid chromatography (RP-HPLC) using either ananalytical Zorbax 300SB-C8 RP-HPLC column and an Agilent Technologies1200 series system or a preparative Vydac C8 RP-HPLC column on a BeckmanCoulter System Gold HPLC. Protein samples were loaded in buffer A (0.1%(v/v) trifluoroacetic acid) and eluted with a linear gradient of 0-100%(v/v) buffer B (60% (v/v) acetonitrile in 0.089% (v/v) trifluoroaceticacid. Eluted proteins were detected by monitoring absorbance at 215 nm.Protein peaks were collected and defensins were identified usingSDS-PAGE, immunoblotting and mass spectrometry.

Purification of Plant Defensins from Pichia pastoris

The Pichia pastoris expression system is well-known and commerciallyavailable from Invitrogen (Carlsbad, Calif.; see the supplier's PichiaExpression Manual disclosing the sequence of the pPIC9 expressionvector).

A single pPIC9-NaD1 P. pastoris GS115 colony was used to inoculate 10 mLof BMG medium (described in the Invitrogen Pichia Expression Manual) ina 100 mL flask and was incubated overnight in a 30° C. shaking incubator(140 rpm). The culture was used to inoculate 500 mL of BMG in a 2 Lbaffled flask which was placed in a 30° C. shaking incubator (140 rpm).Once the OD600 reached 2.0 (˜18 h), cells were harvested bycentrifugation (2,500×g, 10 min) and resuspended into 1 L of BMM medium(OD600 =1.0) in a 5 L baffled flask and incubated in a 28° C. shakingincubator for 3 days. The expression medium was separated from cells bycentrifugation (4750 rpm, 20 min) and diluted with an equal volume of 20mM potassium phosphate buffer (pH 6.0). The medium was adjusted to pH6.0 with NaOH before it was applied to an SP Sepharose column (1 cm×1cm, Amersham Biosciences) pre-equilibrated with 10 mM potassiumphosphate buffer, pH 6.0. The column was then washed with 100 mL of 10mM potassium phosphate buffer, pH 6.0 and bound protein was eluted in 10mL of 10 mM potassium phosphate buffer containing 500 mM NaCl. Elutedproteins were subjected to RP-HPLC using a 40 minute linear gradient asdescribed herein below. Protein peaks were collected and analyzed bySDS-PAGE and immunoblotting with the appropriate anti-defensinantibodies. Fractions containing defensin were lyophilized andresuspended in sterile milli Q ultrapure water. The proteinconcentration of Pichia-expressed defensin was determined using thebicinchoninic acid (BCA) protein assay (Pierce Chemical Co.) with bovineserum albumin (BSA) as the protein standard.

Cell Lines and Culture

Mammalian cell lines used in this study are as follows: human leukemiamonocyte lymphoma U937 cells and human transformed cervical cancer HeLacells. The cells are grown in tissue culture flasks at 37° C. under ahumidified atmosphere of 5% CO₂/95% air, and sub-cultured routinely twoto three times a week according to the rate of proliferation. Allmammalian cells are cultured in RPMI-1640 medium (Invitrogen)supplemented with 10% heat-inactivated fetal bovine serum (FBS,Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 μg/mL streptomycin(Invitrogen). Adherent cell lines are detached from the flask by adding3-5 mL of a mixture containing 0.25% trypsin and 0.5 μM EDTA(Invitrogen).

Cell Viability and Membrane Permeability Assays

Unless otherwise stated, cells are re-suspended at a cell concentrationof 4×10⁵ cells/mL in complete culture RPMI-1640 medium supplemented with10% FBS, 100 μ/mL penicillin and 100 μg/mL streptomycin, and are addedto either a V-bottom 96-well plate or microfuge tubes. Cells are kept at37° C., unless otherwise stated, during protein addition (5 μL) atvarious concentrations or the set concentration of 10 μM. Typically,cells are mixed with the heterogeneous defensin of interest and areincubated at 37° C. for 30 min. In certain experiments, cells are alsoincubated at either 4° C. or 37° C. for 2-60 min prior to flow cytometryanalysis. Cells are added to an equal volume of complete culture mediumcontaining 2 μg/mL propidium iodide (PI, Annexin V-FITC ApoptosisDetection Kit, Invitrogen) and are analysed immediately by flowcytometry using a FACSCanto cell sorter (Becton Dickson, Fanklin Lakes,N.J.) and Cell Quest Pro Software (Becton Dickson). Typically,5000-10000 events per sample are collected and the resultant data isanalysed using FlowJo software (Tree Star, Ashland, Oreg.). Cells aregated appropriately based on forward scatter (FSC) and side scatter(SSC), with the viable cells is determined by their ability to excludePI. For analysis purposes, all data is standardised relative to control(normal cell % ranged from approx. 0-7%).

FITC-dextran binding assays are performed as per the PI uptake assay,with the exception that 100 μg ml⁻¹ of FITC-dextran (Sigma-Aldrich) ispresent during the assay and samples are washed twice with PBScontaining 0.1% BSA prior to addition of 7AAD (1 μg ml⁻¹) and flowcytometry analysis.

MIT Cell Viability Assays

Tumour cells are seeded in quadruplicate into wells of a flat-bottomed96-well microtitre plate (50 μL) at various densities starting at 2×10⁶cells/mL. Four wells containing complete culture medium alone areincluded in each assay as a background control. The microtitre plate areincubated overnight at 37° C. under a humidified atmosphere containing5% CO₂/95% air, prior to the addition of complete culture medium (100μL) to each well and further incubated at 37° C. for 48 h. Optimum celldensities (30-50% confluency) for cell viability assays are determinedfor each cell line by light microscopy.

Tumour cells are seeded in a 96-well microtitre plate (50 μL/well) at anoptimum density determined in the cell optimisation assay as above.Background control wells (n=8) containing the same volume of completeculture medium were included in the assay. The microtitre plate areincubated overnight at 37° C., prior to the addition of proteins atvarious concentrations and the plate is incubated for a further 48 h.The cell viability3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,Sigma-Aldrich) assay is as follows: the MTT solution (1 mg/mL) is addedto each well (100 μL) and the plate is incubated for 2-3 h at 37° C.under a humidified atmosphere containing 5% CO₂/95% air. Subsequently,for adherent cell lines, the media is removed and is replaced withdimethyl sulfoxide (100 μL, DMSO, Sigma-Aldrich), and placed on a shakerfor 5 min to dissolve the tetrazolium salts. In the case of suspensioncells, prior to the addition of DMSO the cells are spun at 1500 rpm for5 min. Absorbance of each well is measured at 570 nm and the IC₅₀ values(the protein concentration to inhibit 50% of cell growth) is determinedusing the Origin Software Program.

Confocal Laser Scanning and Transmission Electron Microscopy

Live imaging was performed on a Zeiss LSM-510 confocal microscope usinga 40× oil immersion objective in a 37° C./5% CO₂ atmosphere. Adherentcells were cultured on coverslips prior to imaging while non-adherentcells were immobilized onto 10% poly-L-lysine-coated coverslips. Allcell types were prepared for imaging in RPMI medium containing 0.1% BSAand 1-2 mg ml⁻¹ PI. NaD1, BODIPY-NaD1, FITC-Dextran (100 μg ml⁻¹) wasadded directly to imaging chamber via capillary tube. In certainexperiments, cells were either, stained with PKH67 (Sigma-Aldrich) ortransfected with plasmid construct for free GFP or GFP-PH(PLCδ) usingLipofectamine™ 2000 Reagent (Invitrogen) as per manufacturer'sinstructions prior to imaging. Transmission electron microscopy (TEM)imaging was performed according to a previously described procedure(Adda et al. 2009).

Lipid Binding Assays

Membrane Lipid Strips™, PIP Strips™ and Sphingo Strips™ (EchelonBiosciences, Salt Lake City, Utah) were incubated with PBS/3% BSA for1-2 h at RT to block non-specific binding. The membrane strips were thenincubated with NaD1 (0.12 μM) diluted in PBS/1% BSA overnight at 4° C.,prior to thorough washing for 60 min at RT with PBS 10.1% Tween-20.Membrane-bound protein was detected by probing the membrane strips witha rabbit anti-NaD1 polyclonal antibody (diluted 1:2000 with PBS/1% BSA)for 1 h at 4° C., followed by a HRP-conjugated donkey anti-rabbit IgGantibody (diluted 1:2000 with PBS/1% BSA) for 1 h at 4° C. After eachantibody incubation, the membrane strips were washed extensively for 60min at RT with PBS/0.1% Tween-20. Chemiluminescence was detected usingthe enhanced chemiluminescence (ECL) western blotting reagent (GEHealthcare BioSciences, NSW Australia) and exposed to Hyperfilm (GEHealthcare BioSciences, NSW, Australia) and developed using an Xomat(All-Pro-Imaging).

Densitometry analysis was performed on images obtained from lipid stripsusing ImageJ (National Institute of Health, Bethesda, Md.). Briefly,circles of equivalent size were traced around areas of interest. Abackground circle of equal size was also placed in the area on themembrane where there is no lipid and set as the background. The areas ofinterest were quantified as the average pixel intensity subtracted fromthe background. Generation of liposomes and liposome pull-downexperiments were conducted using a method as described previously (Zhanget al. 2001; Patki et al. 1997).

Cross-Linking Studies

Protein samples at 1 mg ml⁻¹ (2.5 μl) were incubated with 0.5 mM PIP2 orPA (2.5 μl) at room temperature for 30 min. Protein complexes werecross-linked through primary amino groups by the addition of 12.5 mMbis[sulfosuccinimidyl] suberate (BS³; 5 μl) at room temperature for 30min. Samples were reduced and denatured, and subjected to SDS-PAGE.

Crystallographic Methods

NaD1, purified by RP-HPLC, was lyophilised and resuspended in sterilewater to a final concentration of 15 mg ml⁻¹. Crystals were grown insitting drops at 20° C. in 20% PEG 1500 and 10%succinate-phosphate-glycine buffer pH 9 (Newman et al. 2004). Twocrystal forms were obtained, the first (form A) belonging to space groupP2₁ with a=32.697 Å, b=32.685 Å, c=41.977 Å, α=90°, β=100.828°, γ=90°and the second (form B) belonging to P3₂21 with a=b=33.091 Å, c=128.77Å, α=β=90°, γ=120°. The asymmetric units in both forms contain two NaD1molecules. Diffraction data were collected from crystals flash frozen inmother liquor supplemented with 10% ethylene glycol at 100 K at theAustralian Synchrotron (beamline 3ID1) and processed with XDS (Kabsch2010). For NaD1 crystal form A a heavy atom derivative was obtained bysoaking crystals in mother liquor supplemented with 0.2 M5-amino-2,4,6-triiodoisophthalic acid (I3C) (Beck et al. 2008) for 5min. I3C sites were found with SheIX and refined using Phenix (Adams etal. 2010). Clear and continuous electron density was obtained forresidues the entire molecule. NaD1 crystal form B was solved bymolecular replacement using PHASER (Storoni et al. 2004). The finalmodel for both crystal forms was built with Coot (Emsley and Cowtan2004), refined with Phenix to a resolution of 1.4 Å (form A) and 1.6(form B).

The NaD1:PIP2 complexes were generated by mixing NaD1 at 10 mg ml⁻¹ andPIP2 at a molar ration of 1:1.2. Crystals were grown in sitting drops at20° C. in 0.2 M ammonium sulfate, 7% PEG 3350, 32% MPD and 0.1 Mimidazole pH 7. The structure was solved by molecular replacement withPHASER using the NaD1 structure as a search model. The final model wasbuilt with Coot (Emsley and Cowtan 2004) and refined with Phenix to aresolution of 1.6 Å. Figures were prepared using PyMol (Kvansakul et al.2010)

Transmission Electron Microscopy (TEM)

Samples (10 μl) were applied to 400 mesh copper grids coated with a thinlayer of carbon for 2 min. Excess material was removed by blotting andsamples were negatively stained twice with 10 μl of a 2% uranyl acetatesolution (w/v; Electron Microscopy Services). The grids were air-driedand viewed using a transmission electron microscope, either a JEOL2000FX operated at 120 kV or a JEOL JEM-2010 operated at 80 kV.

Circular Dichroism Spectrum of rNaD1

To examine whether NaD1 purified from P. pastoris (rNaD1) was correctlyfolded, its far UV circular dichroism (CD) spectrum was recorded andcompared with that of native NaD1. The similarity of the two spectraindicates the structure of rNaD1 was not significantly altered comparedto native NaD1.

PCR Mutagenesis of NaD1

Site directed mutagenesis of NaD1 was carried out using the Phusion(Registered Trademark) site-directed mutagenesis kit (Finnzymes).Oligonucleotide primers phosphorylated at the 5′ end were designed toincorporate the desired mutation. The entire template plasmid(pPIC9-NaD1) was amplified in a PCR reaction of 30 cycles with thefollowing temperature profile; 98° C., 30 s; 55° C., 20 s; 72° C., 4 minwith a final extension cycle of 72° C. for 10 min. The linear PCRproduct was then circularized using T4 DNA Quick Ligase for 5 min at RTand transformed into chemically competent TOP10 cells according to themanufacturer's instructions. Constructs were sequenced using the AOX3′primer to ensure the mutation had been correctly incorporated.

Preparation of Electrocompetent P. pastoris

Electrocompetent P. pastoris GS115 cells (Invitrogen) were prepared asdescribed by Chang et al., Mol Biol Cell 16(10):4941-4953, 2005.Briefly, cells grown overnight in YPD (1% w/v Bacto yeast extract, 2%w/v Bacto peptone extract, and 2% w/v dextrose) were harvested andtreated with YPD containing 10 mM DTT, 25 mM HEPES, pH 8, for 15 min at30° C. with shaking. Cells were washed twice in water and once inice-cold 1 M sorbitol, before they were resuspended in 1 M sorbitol anddivided into 80 μL aliquots for storage at −80° C.

Transformation of P. pastoris GS115 with pPIC9 Constructs

Single E. coli TOP10 colonies transformed with each pPIC9 construct areused to inoculate 10 mL of LB containing 100 μg/mL ampicillin andincubated overnight at 37° C. in a shaking incubator. Plasmid DNA isisolated using the Qiaprep (Registered Trademark) miniprep kit (Qiagen)and linearized overnight using the restriction enzyme SalI. Competent P.pastoris GS115 cells (80 μL) are thawed on ice and 1 μg of linearizedDNA is added in an ice-cold Gene Pulser (Registered Trademark)electroporation cuvette with a 0.2 cm gap. DNA is introduced byelectroporation at 1.5 kV, 25 μF, 400Ω (Gene Pulser, Bio-RadLaboratories). Ice-cold 1 M sorbitol (1 mL) is added to the cells beforethey are plated onto MD plates (1.34% w/v yeast nitrogen base, withoutamino acids and with ammonium sulfate [US Biological, YNB], 4×10⁻⁵% w/vbiotin, 2% w/v dextrose) and incubated at 30° C. for 5 days. Positivecolonies are then selected and re-plated onto fresh MD plates.

Generation of a Heterogeneous Plant Defensin

DNA encoding the mature protein of a Class I defensin, for example, NaD2(SEQ ID NO: 27), DmAMP1 (SEQ ID NO: 23) or γ1-H (SEQ ID NO: 25), asbackbone are modified to incorporate a region comprising a loop 5regions (for example NaD1 equivalent residues 34-40) derived from aClass II Solanaceous plant defensin. Exemplary loop 5 regions that areincorporated into the backbone sequence include the following sequences(i) SKILRR, (SEQ ID NO: 2) (ii) SKLLRR, (SEQ ID NO: 4) (iii) SKILRK (SEQID NO: 6), (iv) SKVLRR (SEQ ID NO: 8), (v) SKVLRK(SEQ ID NO: 10), (vi)SKLQRK(SEQ ID NO: 12), (vii) SKLLRN(SEQ ID NO: 14), (viii) SKLLRK(SEQ IDNO: 16), (ix) SKIQRN(SEQ ID NO: 18), (x) RKLQRK (SEQ ID NO: 20) or (xi)KILRR(SEQ ID NO: 89), (xii) KLLRR(SEQ ID NO: 91), (xiii) KILRK(SEQ IDNO: 93), (xiv) KVLRR(SEQ ID NO: 95), (xv) KVLRK(SEQ ID NO: 97), (xvi)KLQRK(SEQ ID NO: 99), (xvii) KLLRN(SEQ ID NO: 101), (xviii) KLLRK(SEQ IDNO: 103), (xix) KIQRN (SEQ ID NO: 105) or (xx) KLQRK(SEQ ID NO: 107).Heterogenous defensin sequences with loop 5 regions as hereindescribedare generated by GenScript USA Inc (Piscataway, N.J.).

The heterogeneous defensin DNA sequences incorporate additional DNAsequences at the 5′ end encoding the amino acids Leu-Glu-Lys-Arg-Ala andincluding a XhoI restriction endonuclease site (CTCGAG) and the KEX2cleavage site at the start of the protein. At the 3′ end, DNA isincluded encoding a stop codon and a NotI restriction endonuclease site.The restriction sites are added so that the genes are subclonable intothe equivalent sites in the pPIC9 expression vector (Invitrogen).

The cloned DNA sequence in the pPIC9 vector, plasmid DNA is isolatedusing a miniprep kit (Invitrogen). The DNA is linearized overnight usingthe restriction enzyme SalI. Electrocompetent P. pastoris GS115 cells(Invitrogen) are prepared as described by Chang et al. (2005). Briefly,cells grow overnight in YPD (1% Bacto yeast extract, 2% Bacto peptoneextract, and 2% dextrose) cells are harvested and are treated with YPDcontaining 10 mM DTT, 25 mM HEPES, pH 8, for 15 min at 30° C. withshaking. Cells are washed twice in water and once in ice-cold 1 Msorbitol, and are resuspended in 1 M sorbitol and are divided into 80 μLaliquots for storage at −80° C. For transformation of the geneconstructs, the electrocompetent P. pastoris GS115 cells (80 μL) arethawed on ice and 1 μg of linearized DNA is added in an ice-cold GenePulser® electroporation cuvette (Bio-Rad Laboratories) with a 0.2 cmgap. DNA is introduced by electroporation at 1.5 kV, 25 μF, 400Ω (GenePulser, Bio-Rad Laboratories). Ice-cold 1 M sorbitol (1 mL) is added tothe cells and then the cells are plated onto MD plates (1.34% yeastnitrogen base, without amino acids and with ammonium sulfate [USBiological, YNB], 4×10-5% biotin, 2% dextrose) and are incubated at 30°C. for 3 days. Positive colonies are selected and are replated ontofresh MD plates. Plasmids are isolated from colonies using a miniprepkit (Invitrogen). Heterogeneous defensins are expressed using a Pichiapastoris expression system and isolated as described above.

Example 1 Class II Solanaceous Plant Defensin-Induced Permeabilisationof Tumour Cells Involves Membrane Blebbing Example 1 Introduction

It has been shown previously in both PCT/AU2011/000760 and U.S. Ser. No.13/166,960, incorporated herein by reference, that Class II defensinsfrom solanaceous plants selectively kill mammalian tumour cells byinducing membrane permeabilisation. To investigate the mechanism ofaction the inventors examined the change in cell morphology when tumourcells are treated with Class II Solanaceous plant defensin NaD1.

Example 1 Results

Live confocal laser scanning microscopy (CLSM) revealed rapid changes onthe cell surface of NaD1 permeabilised cells and showed the formation oflarge bleb-like structures, with (i) an adherent cancer cell line(HeLa—immortal cells derived from human cervical cancer) formingmultiple blebs of different sizes and (ii) a non-adherent cancer cellline (U937—human leukaemia) forming typically 1 to 2 large blebs (FIG.1A). Moreover, bleb size was frequently larger than the actual cell(diameter>20 μm) and did not retract over a period of 20 minutes. Todetermine whether membrane blebbing occurs prior to, during or followingmembrane permeabilisation, we treated U937 cells with NaD1 in thepresence of propidium iodide (PI) and 4 kDa FITC-dextran to monitor theentry of these molecules into NaD1-sensitive cells (FIG. 1B).FITC-dextran and NaD1 were added at 00:35 min, with FITC-dextran beingexcluded from cells with intact membrane. Bleb formation was firstobserved for the cell located at the centre of the panel at 03:25 min,with PI staining appearing at a specific point at the edge of the bleb.From 03:25 to 04:15 min, PI staining was observed in the bleb and thecytoplasm (possibly staining RNA), with FITC-dextran also entering thecell from the bleb site. At 04:20 min, PI-stained molecules were‘expelled’ out of the cell, possibly at the same region that PI firstentered the bleb (FIG. 1B). These data suggest that (i) small moleculessuch as PI can enter the cell initially at a ‘weakened’ point at themembrane bleb, (ii) the bleb continues to enlarge while PI and 4 kDaFITC-dextran enters, and (iii) intracellular contents are released atthe bleb site, representing cytolysis.

Example 2 NaD1 Induces Membrane Blebbing Through Interacting with PIP2Example 2 Introduction

To further investigate the mechanism of NaD1 action, the binding ofBODIPY-labelled NaD1 to tumour cells was tested.

Example 2 Results

Initially, BODIPY-NaD1 was found to mediate membrane permeabilisation ofU937 of a comparable level to unlabelled NaD1 and bound to both viable(7AAD negative) and permeabilised (7AAD positive) U937 cells, with moreBODIPY-NaD1 bound to membrane-damaged cells (FIG. 2A). These datasuggest that NaD1 can interact with tumor cells prior to membranepermeabilisation and accumulate on NaD1-sensitive cells. We nextdetermined the subcellular localisation of BODIPY-NaD1 on permeabilisedtumor cells and showed accumulation of BODIPY-NaD1 at the membranebleb(s), cytoplasm, nucleolus and possibly at certain cytoplasmicorganelles (FIG. 2B). As we have previously shown that the ligands forNaD1 and other class II defensins from solanaceous plants arephosphoinositides, particularly Ptdlns(4,5)P2 (designated PIP2 asdisclosed PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 incorporatedherein by reference) in together with the observation that PIP2 is a keymediator of cytoskeleton-membrane interactions and its sequestration orenzymatic modification can cause blebbing (Sheetz et al., 2001; Raucheret al., 2000), we then asked whether the binding of NaD1 to PIP2 at theinner leaflet of the plasma membrane could lead to the formation oflarge blebs. HeLa cells overexpressing GFP-PH(PLCδ), which bindsspecifically to PIP2, were treated with NaD1 and showed a marked delayfrom the initiation of blebbing (rapid small membrane blebbing) tomembrane permeabilisation when compared to cells expressing free GFP(FIGS. 2C and 2D). These results suggest that the expression ofGFP-PH(PLCδ) competes with NaD1 for PIP2 binding at the inner leaflet ofthe plasma membrane and interferes with NaD1-induced blebbing.

Example 3 Structure of the NaD1:PIP2 Complex Example 3 Introduction

To gain insight into the NaD1:PIP2 interaction at the atomic level,crystal structures of NaD1 on its own as well as in complex with PIP2were determined. The structure of NaD1 was solved using singleisomorphous replacement with anomalous scattering using crystals soakedwith I3C and refined to a resolution of 1.4 Å with a value ofR_(work)/R_(free) of 0.19/0.137.

Example 3 Results

The structure of NaD1 in isolation is identical to our previously solvedNMR structure (FIG. 3A) (Lay et al. 2003b). The monomeric NaD1 model wasthen used to solve the structure of a NaD1:PIP2 complex by molecularreplacement, and we build a model containing 14 molecules of NaD1 and 14PIP2 molecules in the asymmetric unit. The final NaD1:PIP2 complex wasrefined to a resolution of 1.6 Å with a value of R_(work)/R_(free) of0.155/0.184.

Upon PIP2 binding NaD1 forms an arch composed of 14 NaD1 molecules, witha final arch diameter of 90 Å and a width of 35 Å. 14 PIP2 molecules arebound in an extended binding groove on the inside of the arch (FIG. 3B).The entire oligomeric complex is held together by a complex network ofinteractions, which include numerous NaD1:NaD1 as well as NaD1:PIP2interactions (FIG. 3C). The assembly of the oligomer indicates twodistinct interfaces. The first interface is formed by an anti-parallelalignment of beta-strand 1 from two NaD1 molecules (monomers I and II)and exhibits two-fold symmetry between the associated monomers (FIG.3C). It comprises an average buried surface area of 430 Å² and is formedby a network of six hydrogen bonds involving R1, K4, E6, E27, K45 andC47. A second interface is formed by the dimeric NaD1 (comprisingmonomers I and II) and adjacent NaD1 monomers III and IV (FIG. 3C). Thisinterface is formed by hydrogen bonds involving N8 of monomer I, R1, E2,K17 and D31 of monomer II, R1, K17 and D31 of monomer III and N8 ofmonomer IV, effectively forming a dimer of dimers. The full 14-mer isthus constructed using two different interfaces. In addition toNaD1:NaD1 interactions, oligomer formation requires the presence ofPIP2. NaD1 binds PIP2 in a distinct binding site formed by K4 togetherwith residues 33-40, which comprise a characteristic KILRR motif (FIG.3C). PIP2 forms a dense network of hydrogen bonds involving K4, H33,K36, I37, L38 and R40 of a single NaD1 monomer. In oligomeric NaD1:PIP2,a single PIP2 binding site also contains interactions with neighbouringNaD1 monomers (FIG. 3C). Bound PIP2 forms additional hydrogen bonds withR40 from monomer II and K36 from monomer IV′, with the full PIP2 bindingsite in the oligomer comprising contributions from three different NaD1molecules (FIG. 3C). Consequently oligomer formation appears to behighly cooperative, with multiple interactions between adjacent NaD1 andPIP2 molecules required to form the observed 14-mer (FIG. 3D).

Example 4 NaD1:PIP2 can Form Large Oligomeric Complexes Example 4Introduction

To confirm that oligomer formation of NaD1:PIP2 is not a crystallisationartefact, NaD1:PIP2 complexes formed in solution by TEM were examined.

Example 4 Results

It was observed the formation of long string-like structures only whenboth NaD1 and PIP2 are present (FIG. 4A). Furthermore, using across-linking approach of NaD1 with PIP2 in solution, we have alsoconfirmed the formation of NaD1-PIP2 multimers (FIG. 4B). The formationof NaD1 multimers was lipid specific, with PIP2 (binds strongly to NaD1)mediating efficient multimer formation, as apposed to phosphatidic acid(PA) (binds weakly to NaD1) that mediated very low levels ofmultimerisation (FIG. 4B). As described for NaD1, the class II defensinTPP3 preferentially binds PIP2 over PA (described in PCT/AU2011/000760and U.S. Ser. No. 13/166,960 incorporated herein by reference) was alsoefficiently multimerised by PIP2 and not PA. In contrast, the class Idefensin NaD2, which preferentially binds PA over PIP2-seePCT/AU2011/000760 and U.S. Ser. No. 13/166,960 specifically incorporatedherein by reference, was efficiently multimerised by PA and not PIP2.These data demonstrate the specificity of lipid-mediated multimerisationof defensins. It should also be noted the crosslinking studies indicatethat in the absence of lipid, a proportion of NaD1, NaD2 and TPP3, allappear to exist in dimeric as well as monomeric forms (FIG. 4B).

Example 5 Replacement of NaD1 Loop 5 with that of NaD2 ChangesLipid-Binding Specificity and Abolishes Tumour Cell Killing Example 5Introduction

The NaD1:PIP2 structure suggests that the loop 5 region (residues 35-40)of NaD1 is involved in the binding of PIP2, as this region is involvedin multiple contacts with PIP2 (FIGS. 3C and 3D). As the binding of PIP2by NaD1 is involved in the permeabilisation of tumour cells, theinventors propose that loop 5 of Class II Solanaceous plant defensinsare involved in defensin activity related to the killing of tumourcells. To investigate the involvement of loop 5 of Class II Solanaceousplant defensins in lipid binding and tumour cell killing, the inventorsreplaced selected loops of NaD1 with the equivalent regions of NaD2, aclass I defensin that does not bind PIP2 and does not kill tumour cellsas previously disclosed in PCT/AU2011/000760 and U.S. Ser. No.13/166,960 specifically incorporated herein by reference. Tworecombinant NaD1 defensins were generated where NaD2 residues 25-29(loop 4A, a region of NaD1 not involved in lipid binding) and NaD2residues 35-40 (loop 5, a region of NaD1 that forms the extended lipidbinding groove) were inserted into the corresponding loops of NaD1(termed rNaD1(D2L4A) and rNaD1(D2L5) (FIG. 5A).

Example 5 Results

Strikingly, rNaD1 and rNaD1(D2L4A) but not rNaD1(D2L5) permeabilisedU937 cells (FIG. 5B). In terms of lipid binding, similar to native NaD1,rNaD1 and rNaD1(D2L4A) bound most strongly to PIP2, whereas thespecificity of rNaD1(D2L5) was altered in that it bound most strongly toPA (FIGS. 5C and 5D). These results are consistent with our mechanisticmodel and structure data that preferential binding to PIP2 is key tomediate tumor cell lysis and the specificity of lipid recognitionappears to be largely determined by residues located in loop 5 of NaD1.As the residues that define the loop 5 region of Class II Solanaceousdefensins are highly conserved, it is proposed that the loop 5 region asdefined by being flanked by the fifth and sixth (invariant) cysteine(Cys) amino acid residues of a Class II Solanaceous plant defensin aminoacid sequence may be involved in the binding of phospholipids, includingPIP2, and therefore proliferative disease cytotoxicity, for example,through cell membrane permeabilisation activity.

Example 6 NaD1 Loop 5 is Involved in the Interaction with Lipid Example6 Introduction

To further investigate the involvement of loop 5 of Class II Solanaceousdefensins in lipid binding and tumour cell killing/cell permeabilisationthe inventors replaced residues 35-40 of NaD1 with the equivalentregions of other class I defensins, DmAMP1 (Dahlia merckii),γ1-hordothionin (Hordeum vulgare), RsAFP2 (Raphanus sativus) and VrD1(Vigna radiata), to generate rNaD1-DmAMP1L5, rNaD1-ghordoL5,rNaD1-RsAFP2L5, rNaD1-VrD1L5, respectively (FIG. 6A).

Example 6 Results

In contrast to rNaD1, each of the recombinant NaD1 molecules had greatlyreduced or no ability to permeabilise U937 cells (FIG. 6B) and showed apreference for binding to PA as opposed to PIP2 (FIGS. 6C and 6D). Theseresults further support the proposal that preferential binding to PIP2is involved in tumour cell lysis and the specificity of lipidrecognition may be largely determined by residues located in the ClassII Solanaceous loop 5 region as defined by being flanked by the fifthand sixth invariant cysteine amino acid residues of a Class IISolanaceous plant defensin amino acid sequence.

Example 7 Generating Heterogeneous Plant Defensins Example 7Introduction

Exemplary heterogeneous defensins according to the invention areproduced by introducing a modified loop 5 region, or variant or fragmentthereof, into a backbone amino acid sequence of a defensin, for examplea Class I defensin. The modified loop 5 region may be synthesisedaccording to the following structure or sequence: X₁-X₂-X₃-X₄-X₅-X₆,wherein X₁ is serine or arginine, X₂ is lysine, arginine or histidine,X₃ and X₄ are each a hydrophobic amino acid, X₅ is arginine, lysine orhistidine and X₆ is arginine, lysine, histidine, asparagine.

Exemplary amino acid sequences that may be suitable for introductioninto the backbone sequence to generate a heterogeneous defensin asdescribed herein includes the following range of amino acid sequenceswith corresponding polynucleotide sequences indicated in brackets;

(i) (SEQ ID NO: 2) SKILRR  (SEQ ID NO: 3) (tcc aag att ttg aga aga);(ii) (SEQ ID NO: 4) SKLLRR (SEQ ID NO: 5) (tcc aag att ttg aga aga);(iii) (SEQ ID NO: 6) SKILRK  (SEQ ID NO: 7) (tcc aag att ttg aga aag);(iv) (SEQ ID NO: 8) SKVLRR  (SEQ ID NO: 9) (tcc aag gtc ttg aga aga);(v) (SEQ ID NO: 10) SKVLRK  (SEQ ID NO: 11) (tcc aag gtc ttg aga aag);(vi) (SEQ ID NO: 12) SKLQRK  (SEQ ID NO: 13) (tcc aag ttg caa aga aag);(vii) (SEQ ID NO: 14) SKLLRN  (SEQ ID NO: 15) (tcc aag ttg ttg aga aat);(viii) (SEQ ID NO: 16) SKLLRK  (SEQ ID NO: 17)(tcc aag ttg ttg aga aag); (ix) (SEQ ID NO: 18) SKIQRN  (SEQ ID NO: 19)(tcc aag att caa aga aat); (x) (SEQ ID NO: 20) RKLQRK  (SEQ ID NO: 22)(aga aag ttg caa aga aag); (xi) (SEQ ID NO: 89) KILRR  (SEQ ID NO: 90)(aag att ttg aga aga); (xii) (SEQ ID NO: 91) KLLRR  (SEQ ID NO: 92)(aag att ttg aga aga); (xiii) (SEQ ID NO: 93) KILRK  (SEQ ID NO: 94)(aag att ttg aga aag); (xiv) (SEQ ID NO: 95) KVLRR  (SEQ ID NO: 96)(aag gtc ttg aga aga); (xv) (SEQ ID NO: 97 KVLRK  (SEQ ID NO: 98)(aag gtc ttg aga aag); (xvi) (SEQ ID NO: 99) KLQRK   (SEQ ID NO: 100)(aag ttg caa aga aag); (xvii) (SEQ ID NO: 101) KLLRN (SEQ ID NO: 102)(aag ttg ttg aga aat); (xviii) (SEQ ID NO: 103) KLLRK  (SEQ ID NO: 104)(aag ttg ttg aga aag); (xix) (SEQ ID NO: 105) KIQRN (SEQ ID NO: 106)(aag att caa aga aat );  or (xx) (SEQ ID NO: 107) KLQRK (SEQ ID NO: 108) (aag ttg caa aga aag).For example, to produce a heterogeneous defensin, according to theinvention, one of the exemplary loop 5 region sequences (i) to (xx) isintroduced as a substitute loop 5 region into, for example a Class Idefensin amino acid sequence. Exemplary Class I defensin sequences thatare modified to produce a heterogeneous defensin include NaD2 ((SEQ IDNO: 27 and 28), Dm-AMP1 (SEQ ID NO: 23 and 24) and γ1-H (SEQ ID NO: 25and 26). The heterogeneous defensins are produced using standardmolecular biology techniques as described above.

For example, Class I defensin Dm-AMP1:

(SEQ ID No: 23) ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACHVRNGKHMCFC YFNC is modified as described herein to produce a heterogeneous defensinaccording to the invention with the following amino acid sequences(corresponding nucleotide sequences in brackets):

(SEQ ID NO: 29) ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKILRRCFCYFNC(SEQ ID NO: 30) (gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctca tggtgcttgttccaagattttgagaagatgtttctgttatttcaattg ttaa) (SEQ ID NO: 31)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLLRRCFCYFNC  (SEQ ID NO: 32)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaagattttgagaagatgtttctgttatttcaattg ttaa) (SEQ ID NO: 33)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKILRKCFCYFNC  (SEQ ID NO: 34)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaagattttgagaaagtgtttctgttatttcaattgtta  a) (SEQ ID NO: 35)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKVLRRCFCYFNC  (SEQ ID NO: 36)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaaggtcttgagaagatgtttctgttatttcaattgttaa)  (SEQ ID NO: 37)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKVLRKCFCYFNC  (SEQ ID NO: 38)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaaggtcttgagaaagtgtttctgttatttcaattgttaa)  (SEQ ID NO: 39)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLQRKCFCYFNC  (SEQ ID NO: 40)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaagttgcaaagaaagtgtttctgttatttcaattgttaa)  (SEQ ID NO: 41)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLLRNCFCYFNC  (SEQ ID NO: 42)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaagttgttgagaaattgtttctgttatttcaattgttaa)  (SEQ ID NO: 43)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLLRKCFCYFNC  (SEQ ID NO: 44)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaagttgttgagaaagtgtttctgttatttcaattgttaa)  (SEQ ID NO: 45)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKIQRNCFCYFNC  (SEQ ID NO: 46)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgttccaagattcaaagaaattgtttctgttatttcaattgttaa)  (SEQ ID NO: 47)ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACRKLQRKCFCYFNC  (SEQ ID NO: 48)(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaatactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggtgcttgtagaaagttgcaaagaaagtgtttctgttatttcaattgttaa) 

In another example, Class I defensin g-1H:

(SEQ ID NO: 25) RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCDGPLRRCK CMRRCis modified as described herein to produce a heterogeneous defensinaccording to the invention with the following amino acid sequences(corresponding nucleotide sequences in brackets):

(SEQ ID NO: 49) RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKILRRCKCMRRC (SEQ ID NO: 50) (agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagattttgagaagatgtaaatgtatgagaagatg ttaa)  (SEQ ID NO: 51)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLLRRCKCMRRC  (SEQ ID NO: 52)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagattttgagaagatgtaaatgtatgagaagatg ttaa)  (SEQ ID NO: 53)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKILRKCKCMRRC  (SEQ ID NO: 54)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagattttgagaaagtgtaaatgtatgagaagatgtta  a) (SEQ ID NO: 55)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKVLRRCKCMRRC  (SEQ ID NO: 56)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaaggtcttgagaagatgtaaatgtatgagaagatgtta a) (SEQ ID NO: 57)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKVLRKCKCMRRC  (SEQ ID NO: 58)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaaggtcttgagaaagtgtaaatgtatgagaagatgtta a) (SEQ ID NO: 59)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLQRKCKCMRRC  (SEQ ID NO: 60)agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagttgcaaagaaagtgtaaatgtatgagaagatgt taa (SEQ ID NO: 61)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLLRNCKCMRRC  (SEQ ID NO: 62)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagttgttgagaaattgtaaatgtatgagaagatgtta  a) (SEQ ID NO: 63)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLLRKCKCMRRC  (SEQ ID NO: 64)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagttgttgagaaagtgtaaatgtatgagaagatgtta  a) (SEQ ID NO: 65)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKIQRNCKCMRRC  (SEQ ID NO: 66)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgttccaagattcaaagaaattgtaaatgtatgagaagatgttaa)  (SEQ ID NO: 67)RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCRKLQRKCKCMRRC  (SEQ ID NO: 68)(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggtaattgtagaaagttgcaaagaaagtgtaaatgtatgagaagatgttaa) 

In yet another example, Class I defensin NaD2:

(SEQ ID NO: 27) RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCRGFRRRCF CTRPCis modified as described herein to produce a heterogeneous defensinaccording to the invention with the following amino acid sequences(corresponding nucleotide sequences in brackets):

(SEQ ID NO: 69) RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKILRRCFCTRPC (SEQ ID NO: 70) (agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaagggtggcgactgctccaagattttgagaagatgttctgtaccaggccttgctaa)  (SEQ ID NO: 71)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLLRRCFCTRPC  (SEQ ID NO: 72)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaagattttgagaagatgtttctgtaccaggccttg ctaa) (SEQ ID NO: 73)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKILRKCFCTRPC  (SEQ ID NO: 74)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaagatttgagaaagtgtttctgtaccaggccttgc taa) (SEQ ID NO: 75)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKVLRRCFCTRPG  (SEQ ID NO: 76)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaaggtcttgagaagatgtttctgtaccaggccttg  ctaa) (SEQ ID NO: 77)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKVLRKCFCTRPC  (SEQ ID NO: 78)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaaggtcttgagaaagtgtttctgtaccaggccttg ctaa) (SEQ ID NO: 79)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLQRKCFCTRPC  (SEQ ID NO: 80)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaagttgcaaagaaagtgtttctgtaccaggccttg  ctaa) (SEQ ID NO: 81)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLLRNCFCTRPC  (SEQ ID NO: 82)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaagttgttgagaaattgtttctgtaccaggccttg  ctaa) (SEQ ID NO: 83)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLLRKCFCTRPC  (SEQ ID NO: 84)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaagttgttgagaaagtgtttctgtaccaggccttg ctaa) (SEQ ID NO: 85)RTCESGSHRFKGPCARDSNCATVCLTEGFSGGDCSKIQRNCFCTRPC (SEQ ID NO: 86)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgctccaagattcaaagaaattgtttctgtaccaggccttg ctaa) (SEQ ID NO: 87)RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCRKLQRKCFCTRPC  (SEQ ID NO: 88)(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaagagatagcaactgtgccaccgtctgtttgacagaaggattttccggtggcgactgcagaaagttgcaaagaaagtgtttctgtaccaggccttg ctaa)

Example 7 Results

Using the methods described above, the resulting heterogeneous defensinsare tested for lipid binding specificity and the ability to kill tumourcells by permeabilisation. It is expected that the heterogeneousdefensins display the ability of any one or more of inter alia, bindingof PIP2, ability to permeabilise tumour cells.

Example 8 Single Amino Acid Changes in Class II Solanaceous DefensinNaD1 Loop 5 Abolish Killing of Tumour Cells Example 8 Introduction

To investigate the role of amino acids K36 and R40 in loop 5 of theClass II Solanaceous defensin NaD1 for tumour cell killing, each ofthese residues were replaced with an amino acid of the opposite charge,glutamic acid (E), to generate K36E and R40E mutants.

Example 8 Results

In contrast to rNaD1, the K36E and R40E mutant NaD1 molecules hadgreatly reduced ability to permeabilise U937 cells (FIG. 8). Theseresults further support the involvement of loop 5 of NaD1 by identifyingtwo residues in this region that appear to be required for tumour cellkilling.

Example 9 In Vitro Anti-Tumour Activity of Heterogeneous Defensin

The effect of the heterogeneous defensin on the viability of tumour celllines and primary human cell isolates is determined using a3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)in vitro cell culture viability assay. The tumour cell lines tested areHCT116 (human colon cancer), MCF-7 (human breast cancer), MM170 (humanmelanoma), PC3 (human prostate cancer), B16-F1 (mouse melanoma), CASMC(human coronary artery smooth muscle cells) and HUVEC (human umbilicalvein endothelial cells). Cells are seeded into 96-well flat-bottomedmicrotitre plates at the following cell numbers: MM170 (2×10⁴/well),MCF-7 (2×10⁴/well), HCT-116 (5×10³/well), PC3 (5×10³/well), B16-F1(2×10³/well), HUVEC (3×10³/well), CASMC (5×10³/well) and are culturedovernight. The heterogeneous defensin is added to cells to finalconcentrations ranging from 1 to 100 μM and is incubated for 48 h, uponwhich MTT assays are carried out as described in the Materials andMethods.

Example 10 Effect of the Heterogeneous Defensin on the Permeabilisationof Human Cell In Vitro

The ability of the heterogeneous defensin to permeabilise tumour cellsis assessed using flow cytometry to determine the uptake of thefluorescent dye propidium iodide (PI) (2 mg/mL) by U937 and MM170 cells(4×10⁵/mL) following the treatment of cells with increasingconcentrations of the heterogeneous defensin (0 to 100 μM) for 30 min.

Those skilled in the art will appreciate that the disclosure describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the disclosureincludes all such variations and modifications. The disclosure alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1. A heterogeneous plant defensin comprising a first polypeptidesequence and a second polypeptide sequence, wherein the secondpolypeptide sequence is derived from a plant defensin other than theplant defensin from which the first polypeptide sequence is derived. 2.The heterogeneous plant defensin of claim 1, further comprising a thirdpolypeptide sequence, wherein the third polypeptide sequence is derivedfrom the same plant defensin as the first polypeptide sequence.
 3. Theheterogeneous plant defensin of claim 1 or 2, comprising the sequence:(SEQ ID NO: 1)X_(A)-C1-X_(B)-C2-X_(C)-C3-X_(D)-C4-X_(E)-C5-X_(F)-C6-X_(G)-C7-X_(H)-C8-X_(I),;

wherein C1, C2, C3, C4, C5, C6, C7 and C8 is cysteine, said cysteinebeing an invariant cysteine, X_(A), X_(B), X_(C), X_(D), X_(E), X_(F),X_(G), X_(H), X_(I) is any naturally occurring amino acid and; X_(A) is1 to 7 amino acids in length; X_(B) is 9, 10 or 11 amino acids inlength; X_(C) is 3 to 8 amino acids in length; X_(D) is 3 amino acids inlength; X_(E) is 9 to 13 amino acids in length; X_(F) is 4, 5, 6, 7 or 8amino acids in length; X_(G) is 1 amino acid in length; X_(H) is 1 to 4amino acids in length; and X_(I) is 0 or 1 amino acid in length.
 4. Theheterogeneous plant defensin of claim 3, wherein the second polypeptidesequence is positioned between the fourth and eighth invariant cysteineamino acids corresponding to the sequence set forth as SEQ ID NO:1. 5.The heterogeneous plant defensin of claim 3, wherein the secondpolypeptide sequence is positioned between the fifth and sixth invariantcysteine amino acids corresponding to the sequence set forth as SEQ IDNO:1.
 6. The heterogeneous plant defensin of any one of claims 1 to 5,wherein the second polypeptide sequence corresponds to an amino acidsequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁ is serine or arginine,X₂ is lysine, arginine or histidine, X₃ and X₄ are each a hydrophobicamino acid, X₅ is arginine, lysine or histidine and X₆ is arginine,lysine, histidine or asparagine
 7. The heterogeneous plant defensin ofany one of claims 1 to 5, wherein the second polypeptide sequencecorresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆,wherein X₁ is serine or arginine, X₂ is lysine, X₃ is isoleucine,leucine or valine, X₄ is leucine or glutamine, X₅ is arginine and X₆ isarginine, lysine or asparagine.
 8. The heterogeneous plant defensin ofany one of claims 1 to 5, wherein the second polypeptide sequencecorresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, whereinX₂ is lysine, arginine or histidine, X₃ and X₄ are each a hydrophobicamino acid, X₅ is arginine, lysine or histidine and X₆ is arginine,lysine, histidine or asparagine.
 9. The heterogeneous plant defensin ofany one of claims 1 to 5, wherein the second polypeptide sequencecorresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, whereinX₂ is lysine, X₃ is isoleucine, leucine or valine, X₄ is leucine orglutamine, X₅ is arginine and X₆ is arginine, lysine or asparagine. 10.The heterogeneous plant defensin of any one of claims 1 to 9, whereinthe second polypeptide sequence corresponds to an amino acid sequenceselected from the group comprising SEQ ID NO:29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
 87. 11. Theheterogeneous plant defensin of claim 10, wherein the second polypeptidesequence corresponds to an amino acid sequence comprising S-K-I-L-R-R(SEQ ID NO: 2).
 12. The heterogeneous plant defensin of claim 10,wherein the second polypeptide sequence corresponds to an amino acidsequence comprising K-I-L-R-R (SEQ ID NO: 89).
 13. The heterogeneousplant defensin of any one of claims 1 to 12, wherein the firstpolypeptide sequence is derived from a Class I defensin.
 14. Theheterogeneous plant defensin of any one of claims 1 to 13, wherein thesecond polypeptide sequence is derived from a Class II Solanaceous plantdefensin.
 15. The heterogeneous plant defensin of any one of claims 1 to14, further comprising one or more amino acid substitutions, deletionsor additions.
 16. The heterogeneous plant defensin of any one of claims1 to 15, further comprising one or more amino acids selected from thegroup consisting of: K (Lysine) at or around +1 amino acid residuerelative to the first invariant Cysteine; K (Lysine) at or around +5amino acid residues relative to the fourth invariant Cysteine; D(Aspartic Acid) at or around −3 amino acids residues relative to thefifth invariant Cysteine; H (Histidine) at or around −1 amino acidresidue relative to the fifth invariant Cysteine; and/or K (Lysine) ator around +2 amino acid residues relative to the seventh invariantCystine, and/or conservative substitutions, functional and/or structuralequivalents thereof.
 17. The heterogeneous plant defensin of any one ofclaims 1 to 16, wherein the second polypeptide is derived from a ClassII Solanaceous plant defensin from Nicotiana spp., Petunia spp., Solanumspp., or Capsicum spp.
 18. The heterogeneous plant defensin of any oneof claims 1 to 17, wherein the second polypeptide is derived from NaD1,NsD1, NsD2, NoD173, TPP3, PhD1, PhD1A, PhD2, TPP3, FST, NeThio1,NeThio2, NpThio1, Na-gth or CcD1.
 19. The heterogeneous plant defensinof any one of claims 1 to 18, wherein the first polypeptide is derivedfrom a Class I plant defensin from Nicotiana spp., Hordeum spp., Pisumspp., Medicago, Dahlia spp., Raphanus spp or Zea spp.
 20. Theheterogeneous plant defensin of any one of claims 1 to 19, wherein thefirst polypeptide is derived from the Class I defensin NaD2, g1-H, Psd1,Ms-Def1, Dm-AMP1, R5-AFP2 or g-zeathionin2.
 21. The heterogeneous plantdefensin of any one of claims 1 to 20, comprising the amino acidsequence set forth as any one of SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ IDNO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
 87. 22. Theheterogeneous plant defensin of any one of claims 1 to 20, encoded bythe nucleic acid sequence set forth as any one of SEQ ID NO:30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88.23. The heterogeneous plant defensin of any one of claims 3 to 20,comprising a serine amino acid positioned adjacent to the fifthinvariant cysteine amino acid corresponding to the sequence set forth asSEQ ID NO:1, wherein the serine amino acid is positioned to theC-terminal side of the fifth invariant cysteine amino acid residue. 24.The heterogeneous plant defensin of any one of claims 1 to 23, whereinthe heterogeneous plant defensin displays enhanced anti-proliferativedisease and/or cytotoxic activity relative to the defensin from whichthe first polypeptide sequence is derived.
 25. The heterogeneous plantdefensin of any one of claims 1 to 24, wherein the heterogeneousdefensin binds to a phospholipid.
 26. The heterogeneous plant defensinof any one of claims 1 to 24, wherein the second polypeptide sequence,or part thereof, binds to a phospholipid.
 27. The heterogeneous plantdefensin of any one of claims 1 to 24, wherein the heterogeneousdefensin binds to phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2(PIP2).
 28. The heterogeneous plant defensin of any one of claims 1 to24, wherein the second polypeptide sequence, or part thereof, binds tophosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2 (PIP2).
 29. Theheterogeneous plant defensin of claim 25 or 26, wherein the phospholipidis located in a cell membrane.
 30. The heterogeneous plant defensin ofclaim 27 or 28, wherein phosphatidylinositol 4,5-bisphosphate orPtdlns(4,5)P2(PIP2) is located in a cell membrane.
 31. The heterogeneousplant defensin of any one of claims 1 to 30, wherein the heterogeneousplant defensin has cell membrane permeabilisation activity.
 32. Theheterogeneous plant defensin of any one of claims 29 to 31, wherein thecell membrane is a tumour or cancer cell membrane.
 33. A nucleic acidencoding the heterogeneous plant defensin of any one of claims 1 to 32,or a functional fragment thereof.
 34. A heterogeneous plant defensinencoded by the nucleic acid of claim
 33. 35. A vector comprising thenucleic acid of claim
 34. 36. A host cell comprising the vector of claim35.
 37. A heterogeneous plant defensin produced by the host cell ofclaim
 36. 38. A pharmaceutical composition comprising the heterogeneousplant defensin of any one of claim 1 to 32, 34 or 37, the nucleic acidof claim 33, the vector of claim 35, or the host cell of claim 36,together with a pharmaceutically acceptable carrier, diluent orexcipient.
 39. A method for preventing or treating a proliferativedisease, wherein the method comprises administering to a subject atherapeutically effective amount of the heterogeneous plant defensin ofany one of claim 1 to 32, 34 or 37, the nucleic acid of claim 33, thevector of claim 35, or the host cell of claim 36 or the pharmaceuticalcomposition of claim 38, thereby preventing or treating theproliferative disease.
 40. The method according to claim 39, wherein theproliferative disease is cancer.
 41. The method according to claim 40wherein the cancer is selected from the group comprising basal cellcarcinoma, bone cancer, bowel cancer, brain cancer, breast cancer,cervical cancer, leukemia, liver cancer, lung cancer, lymphoma,melanoma, ovarian cancer, pancreatic cancer, prostate cancer or thyroidcancer.
 42. Use of the heterogeneous plant defensin of any one of claim1 to 32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35,or the host cell of claim 36 or the pharmaceutical composition of claim38 in the preparation of a medicament for preventing or treating aproliferative disease.
 43. A kit for preventing or treating aproliferative disease, wherein the kit comprises a therapeuticallyeffective amount of the heterogeneous plant defensin of any one of 1 to32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35, orthe host cell of claim 36 or the pharmaceutical composition of claim 38.44. Use of the kit of claim 43 for preventing or treating aproliferative disease, wherein the therapeutically effective amount ofthe heterogeneous plant defensin of any one of 1 to 32, 34 or 37, thenucleic acid of claim 33, the vector of claim 35, or the host cell ofclaim 36 or the pharmaceutical composition of claim 38 is administeredto a subject, thereby preventing or treating the proliferative disease.45. The use of claim 42 or 44, wherein the proliferative disease iscancer.
 46. The use of claim 45 wherein the cancer is selected from thegroup comprising basal cell carcinoma, bone cancer, bowel cancer, braincancer, breast cancer, cervical cancer, leukemia, liver cancer, lungcancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostatecancer or thyroid cancer.
 47. A heterogeneous plant defensin of any oneof claims of any one of claim 1 to 32, 34 or 37 when used for preventingor treating a proliferative disease.
 48. The heterogeneous plantdefensin of claim 47, wherein the proliferative disease is cancer. 49.The heterogeneous plant defensin of claim 48 wherein the cancer isselected from the group comprising basal cell carcinoma, bone cancer,bowel cancer, brain cancer, breast cancer, cervical cancer, leukemia,liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer,pancreatic cancer, prostate cancer or thyroid cancer.